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WO2008109757A2 - Procédés et compositions mettant en œuvre des protéines hybrides d'immunoglobuline polymériques - Google Patents

Procédés et compositions mettant en œuvre des protéines hybrides d'immunoglobuline polymériques Download PDF

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Publication number
WO2008109757A2
WO2008109757A2 PCT/US2008/056066 US2008056066W WO2008109757A2 WO 2008109757 A2 WO2008109757 A2 WO 2008109757A2 US 2008056066 W US2008056066 W US 2008056066W WO 2008109757 A2 WO2008109757 A2 WO 2008109757A2
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Prior art keywords
polypeptide
antigen
region
hch2
seq
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WO2008109757A3 (fr
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Barry G. Arnason
Mark A. Jensen
David M. White
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Iterative Therapeutics Inc
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Iterative Therapeutics Inc
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Priority to EP08731555A priority Critical patent/EP2164859A4/fr
Priority to CA002679743A priority patent/CA2679743A1/fr
Publication of WO2008109757A2 publication Critical patent/WO2008109757A2/fr
Publication of WO2008109757A3 publication Critical patent/WO2008109757A3/fr
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/08—Clostridium, e.g. Clostridium tetani
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001103—Receptors for growth factors
    • A61K39/001104—Epidermal growth factor receptors [EGFR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001103—Receptors for growth factors
    • A61K39/001106—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ErbB4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
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    • A61K39/001111—Immunoglobulin superfamily
    • A61K39/001112—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001169—Tumor associated carbohydrates
    • A61K39/00117—Mucins, e.g. MUC-1
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001169—Tumor associated carbohydrates
    • A61K39/001171—Gangliosides, e.g. GM2, GD2 or GD3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001174—Proteoglycans, e.g. glypican, brevican or CSPG4
    • A—HUMAN NECESSITIES
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    • A61K39/0005—Vertebrate antigens
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    • A61K39/00118—Cancer antigens from embryonic or fetal origin
    • A61K39/001182—Carcinoembryonic antigen [CEA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/0011—Cancer antigens
    • A61K39/00119—Melanoma antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001193—Prostate associated antigens e.g. Prostate stem cell antigen [PSCA]; Prostate carcinoma tumor antigen [PCTA]; PAP or PSGR
    • A61K39/001194—Prostate specific antigen [PSA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6056—Antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates generally to the field of immunology.
  • An early step is Ag internalization by APCs of the innate immune system, notably by dendritic cells (DCs), the most potent APC type, and the one best able to present Ag to naive T cells (Trombetta and Mellman, 2005).
  • Internalized Ag is processed through the endosomal/lysosomal path. Processed peptides, bound to MHC molecules, are then delivered to the cell surface. Those T cells with appropriate receptors respond to such peptides provided co-stimulatory molecules are expressed by the DC.
  • a second signal is often required to drive DC maturation and efficient co-stimulatory molecule expression.
  • Ag activates B cells bearing appropriate surface immunoglobulin directly to produce IgM.
  • CD4 + T cells having responded to processed Ag, induce immunoglobulin class- switching from IgM to IgG.
  • Immune complexes exhibit diverse biological activities; some that contribute to disease whereas others ameliorate disease.
  • Deposition of IgG containing IC on tissue surfaces, as for example in glomeruli, can contribute to the pathogenesis of antibody- mediated autoimmune diseases.
  • IC can favorably modulate T- and B- cell activation pathways via binding to Fc receptors expressed on immunocytes.
  • Aggregated IgG (AIG) shares some features and biological activities with IC.
  • Monomelic IgG can ameliorate disease progression in animal models of autoimmune disease (Miyagi et al, 1997; Gomez-Guerrero et al, 2000).
  • Monomeric IgG can be used therapeutically, usually in massive doses, to treat antibody-mediated diseases in man.
  • the protective effect in antibody-mediated diseases may be achieved in part through blockade of Fc ⁇ Rs such that binding of IC to them is impeded (Clynes et al, 1998).
  • IgG administration also favorably affects the course of T- cell mediated autoimmune diseases such as multiple sclerosis (Fazekas et al 1997; Sorensen et al, 1998; Achiron et al, 1998).
  • Aggregated IgG has been proposed as a treatment for autoimmune diseases of humans.
  • the use of aggregated IgG has been studied as a treatment for multiple sclerosis and other autoimmune diseases.
  • aggregated IgG has major limitations. IgG is commonly aggregated by exposure to heat; the resultant aggregates are bound together in a random fashion limiting reproducibility from one preparation to the next. Preparations contain a heterogeneous collection of aggregates of varying size in diverse conformations.
  • U.S. Patents 5,714,147 and 5,455,165 disclose some hybrid immunoglobulin molecules and the expression vectors encoding them. These chimeric molecules can improve the circulating plasma half-life of ligand binding molecules, and can comprise a lymphocyte homing receptor fused to an immunoglobulin constant region.
  • U.S. Patent 6,046,310 discloses FAS ligand fusion proteins comprising a polypeptide capable of specifically binding an antigen or cell surface marker for use in treatment of autoimmune disorders.
  • the fusion protein preferably comprises IgG2 or IgG4 isotype, and may comprise antibodies with one or more domains, such as the CH2, CHl or hinge deleted.
  • Majeau et al. (1994) discusses Ig fusion proteins used for the inhibition of T cell responses. These fusion proteins comprise IgGl and LFA-3.
  • Eilat et al. (1992) disclose a soluble chimeric Ig heterodimer produced by fusing TCR chains to the hinge region, CH2, and CH3 domains of human IgGl.
  • Immunoglobulin fusion proteins can be employed to express proteins in mammalian and insect cells (Ashkenazi, et al., 1997). Fusion protein platforms can permit the introduction of additional functions, for example, inclusion of the amino- terminal CD8 ⁇ domain may result in the co-ligation of FcR on lymphocytes to MHC I on antigen presenting cells (Alcover, et al., 1993; Meyerson, et al, 1996).
  • Ig proteins and variants have also been studied for their therapeutic effect on autoimmune diseases, including a recombinant polymeric IgG that mimics the complement activity of IgM (Smith and Morrison, 1994) where the polymeric IgG is formed by the polymerization of H 2 L 2 subunits.
  • Greenwood et al. (1993) discusses therapeutic potency relative to the structural motifs involving the human IgG antibodies, IgGl, IgG3, and IgG4.
  • U.S. Patent 5,998,166 discloses human Fc ⁇ R -III variants, which can be used in the therapy or diagnosis of autoimmune diseases.
  • Patent 5,830,731 discloses novel expression vectors in which cell surface antigens cloned according to that invention appear to have diagnostic and therapeutic utility in immune-mediated infections.
  • Cell surface antigens that are used to regulate lymphocyte activation appear to achieve antigen aggregation in vitro by incubating lymphocytes with immobilized ligands or antibodies or their fragments (WO9942077).
  • the aggregated IgG and Fc aggregates have limited reproducibility, containing a random and heterogeneous mixture of protein thereby limiting their effectiveness as therapeutic agents.
  • Other problems include a lack of an ability to target a number of cell types with a single agent and size limitations.
  • the inventive polypeptide has an immunoglobulin framework and consists of an Fc region linked to two arms.
  • the Fc region consists of two Fc amino acid chains and each Fc amino acid chain is linked to one of the two arms.
  • Each arm consists of an HCH2 polymer linked to an antigen portion, the HCH2 polymer consists of two to six linear copies of an HCH2 monomer, and the HCH2 monomer consists of at least a fragment of an HCH2 region.
  • At least a fragment of an HCH2 region includes a hinge region; and at least one hinge region cysteine of the HCH2 monomer is mutated to serine.
  • the inventive polypeptide can consist of two amino acid chains where each amino acid chain consists of (a) an Fc portion which includes the C-terminus of the amino acid chain; (b) a polymer portion consisting of two to six linear copies of an HCH2 monomer; and (c) an antigen portion which includes the N-terminus of the amino acid chain.
  • the N-terminus of the Fc portion is linked to the C-terminus of the polymer portion, and the N-terminus of the polymer portion is linked to the C- terminus of the antigen portion.
  • the two amino acid chains are linked using one or more disulfide bonds located in the Fc portion of each amino acid chain.
  • the HCH2 monomer can consist of at least a fragment of an HCH2 region, wherein the at least a fragment of an HCH2 region includes a hinge region.
  • the inventive polypeptide has Fc amino acid chains selected from the group consisting of: an amino acid chain of the IgGl Fc region, an amino acid chain of the IgG3 Fc region, an amino acid chain of the IgG2a Fc region, SEQ ID NO: 47, SEQ ID NO: 48, and fragments thereof.
  • the inventive polypeptide is capable of binding to Fc ⁇ R or of targeting cells expressing Fc ⁇ R.
  • the inventive polypeptide has the HCH2 region selected from the group consisting of: a human IgGl HCH2 region, a human IgG2 HCH2 region, a human IgG3 HCH2 region, a human IgG4 HCH2 region, a mouse IgG2a, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 50, SEQ ID NO: 51, and fragments thereof.
  • the inventive polypeptide has three hinge region cysteines of the HCH2 monomer mutated to serine.
  • the antigen portion is an antigen or an epitope, which can be a protein or protein fragment, a Botulinum neurotoxin protein or fragment thereof, BoNT/A Hc, BoNT/A HcN, BoNT/A HcC, HSAl, CD8 ⁇ , FABP7, PLP, MBP, PLP- MBP, PLP-PLP, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 43, or fragments thereof.
  • a composition or vaccine comprises the inventive polypeptide.
  • the inventive polypeptide is provided to a subject, used in a vaccine, or used to induce immunity.
  • inventions include methods for making the inventive polypeptides or the nucleic acids used to encode the inventive polypeptides.
  • FIG. 1 Design Rationale. Schematic depicts the design rationale utilized in the construction of the HCH2 polymer, a feature of which is the iterative regeneration of cloning sites in the extension step.
  • the ⁇ HCH2 shown in the schematic represents an HCH2 monomer in which the hinge cysteines have been changed to serines.
  • FIG. 2. Schematic illustrating the structures of IgG, Fc fusion protein,
  • HCH2 polymers R2, R3, and R4. Drawing on the left represents IgG 1 polypeptide.
  • the element labeled Fc represents the IgG 1 framework composed of the hinge, CH2 and CH3 domains of human IgG 1 with one light chain missing to reveal heavy chain structure.
  • the small chain extending from the CH2 region represents N-linked carbohydrate at Asn297.
  • the second drawing depicts an Fc fusion protein wherein an antigen portion (represented as a hexagon in the drawing and labeled as antigen) has been fused to the Fc region.
  • the third drawing shows an HCH2 polymer.
  • the darkened ovals represent 4 repeated hinge region and CH2 domain units.
  • hinge region cysteines were mutated to serines. The mutations leave intact those hinge residues known to interact with Fc ⁇ Rs.
  • the final three drawings show HCH2 polymers with 2, 3, and 4 HCH2 monomers per polymer integrated into Fc fusion protein structure.
  • FIGS. 3A, and 3B Western Blot analyses of Fc, R2, R3 and R4.
  • FIG. 3A Recombinant proteins were separated on 7% SDS-PAGE gels and stained with Coomassie brilliant blue dye to reveal protein.
  • FIG. 3B Recombinant proteins were transferred to nitrocellulose membrane and stained with antibodies directed against human Fc. Note that the human IgG control and the fusion proteins are recognized by anti-Fc antibody.
  • FIG. 4 Western blot Analysis. HSA 1R4 and MSAlmR4 (75 ng) were electrophoresed on 7% SDS-polyacrylamide gels (SDS-PAGE). Gels were either stained with Coomassie Blue to reveal total protein(Panel A) or transferred to nitrocellulose membranes for Western Blot analysis (Panels B and C). Panel A. MSAlmR4 (Lane M) and HSA1R4 (Lane H) were resolved on SDS-PAGE gels and stained to reveal total protein. As expected, the panel shows that MSAlmR4 and HSA 1R4 have similar molecular weights. Panel B.
  • MSAlmR4 and HSA1R4 were run on SDS-PAGE gels and transferred to a nitrocellulose membrane.
  • the membrane was probed with goat anti- mouse IgG2a-HRPO (Caltag) to reveal the presence of proteins with mouse IgG2A sequences. Only MSAlmR4 bound the antibody indicating that MSAlmR4 and HSA 1R4 are antigenically distinct.
  • Panel C The membrane from Panel B. was stripped of detecting antibody and reprobed with goat anti-human Fc-HRPO (Bethyl Labs) to reveal human Fc sequences. Only HSA1R4 bound the detecting antibody again indicating that HSA 1R4 and MSAlmR4 are distinct.
  • FIG. 5 Binding of CIq to HSA1R4 measured by ELISA.
  • HSA1R4, HSAlFc, and monomeric human IgG (Sigma Corp.) were immobilized onto ELISA plates at 2 to 10 ⁇ g/ml.
  • CIq was added to ELISA plates at 4 ⁇ g/ml.
  • Bound CIq was detected using HRP conjugated goat anti-Clq IgG followed by OPD addition. Data are expressed as O.D.
  • Approximately equal amounts of CIq bind HSA1R4, HSAlFc, and monomeric IgG at all concentrations of ligand tested. Data shown are representative of three separate experiments.
  • FIG. 6. R4 ligand binds far more efficiently to Fc ⁇ R than monomeric Ig or Ig fusion proteins.
  • Panels A-E display binding results for five different Fc receptors. Low-affinity Fc ⁇ R were coated onto 96 well plates at 4 ⁇ g/mL, Fc ⁇ RI was at 2 ⁇ g/mL. Plates were washed, blocked, and overlain with the Fc ⁇ R ligands at the indicated concentrations. Plates were washed and bound ligand was detect using HRPO-Protein G which binds all ligands at a single site. Results shown are from a representative assay of four performed.
  • FIG. 7A, 7B, 7C HSA1R4 binds to Fc ⁇ Rs expressed on the surface of living cells. Flow cytometric analysis of HSAl R4 binding detected with FITC anti-HSA goat Ig is shown in black; background fluorescence of cells stained with HSA 1R4 and FITC goat Ig in white.
  • FIG. 7A Pre-incubation with antibody to Fc ⁇ RI (clone 10.1) partially blocks binding of HS A 1R4 to U937 cells (gray).
  • FIG. 7B Preincubation with antibody to Fc ⁇ RII (clone FLI8.26) partially blocks binding of HSA 1R4 to U937 cells (gray).
  • Fig. 7C Preincubation with antibody to both Fc ⁇ RI and Fc ⁇ RII completely blocks binding of HSA1R4 to U937 cells (gray).
  • HSA 1R4 induces greater proliferative responses in PBMC than does HSA 1R3, HSA 1R2, or HSAlFc.
  • PBMC activation with HCH2 polymer proteins correlates directly with the number of HCH2 region repeats indicating a high level of sensitivity of Fc ⁇ receptors to HCH2 number in the HCH2 polymer proteins.
  • 2 x 10 5 freshly isolated PBMC were plated into 96 well plates in the presence of medium alone, or with IL-2 (1 ng/mL) and varying concentrations of HSA 1R4, HSA 1R3, HSA 1R2, or HSAlFc for 72 hr.
  • the graph compares the proliferative response of PBMC to varying dilutions of each HCH2 polymer protein used. CPM is shown on the y-axis and micrograms/ml of HCH2 polymer protein used is shown on the x-axis.
  • the dose response curves show that as the number of HCH2 repeats increases in each ligand so does the efficiency with which it induces PBMC proliferation.
  • HSA1R4 induces significantly greater proliferation by PBMC than does HSA 1R3, HSA 1R2, and HSAlFc at the concentrations indicated on the figure.
  • Data represent the average from four individuals ⁇ SEM.
  • FIG. 9. I. V. injection of HSA1R4 increases HSAl-specific IgG antibody responses in SJL mice.
  • HSA 1R4 increases HSAl-specific IgG antibody responses in SJL mice following i.v. injection of 50 ⁇ g of HSA1R4, HSAlFc, or HSAl.
  • Anti-HSA Ab titers are higher in mice given HSA 1R4 than in mice receiving HSAlFc (p ⁇ 0.001).
  • FIG. 11 HSA1R4 increases HSAl-specific Ab responses in C57BL/6 mice.
  • HSA- reactive IgG mice were immunized with 50 ⁇ g of HSAl R4, HSAlFc, and HSAl subcutaneously in Ribi adjuvant. Sera were obtained two weeks later (7 mice per group). Titers of HSA- reactive IgG are shown as mean ⁇ SEM. HSA-specif ⁇ c Ab titers are 10 fold higher in mice given HSA1R4 than in mice given HSAlFc (p ⁇ 0.05) and 50 fold higher than in mice given HSAl (p ⁇ 0.005).
  • FIG. 12A, 12B FIG. 12A. HSAl -induced T cell proliferation is higher in splenocytes from mice immunized with HSA 1R4 than in splenocytes from mice immunized with HSAlFc (p ⁇ 0.004). Shown are proliferative responses of cells from mice immunized 2 wk previously with HSA 1R4 or HSAlFc in Ribi adjuvant, and challenged in vitro with HSAl. Data shown are the mean ⁇ SEM of four experiments.
  • FIG. 12B HSA1R4 augments presentation of HSAl to HSA-reactive T cells.
  • FIG 13 Schematic of BoNT/A toxin organization. BoNT is expressed as a single chain 150 kD polypeptide which following proteolytic cleavage results in a light chain (-50 kD) linked by disulphide bonds to a heavy chain (-100 kD).
  • BoNT activities map to discrete regions within the polypeptide chains: Endoprotease activity resides within the light chain.
  • the heavy chain is responsible for receptor binding and translocation.
  • the heavy chain can be further subdivided both functionally and proteolytically into an amino-terminal fragment (HN), involved in ion-channel formation and light chain translocation, and a carboxyl-terminal fragment (Hc) involved in receptor binding.
  • Hc fragment is composed of two -200 amino acid sub-domains that are structurally distinct; the amino-terminal portion, HcN (residues 871 to 1078 of the holotoxin) and the carboxyl-terminal portion, HcC (residues 1090 to 1296 of the holotoxin).
  • HcN amino-terminal fragment
  • HcC carboxyl-terminal portion
  • HcR4 antigens bind efficiently to Fc ⁇ R.
  • Panels A-E display binding results for five different Fc receptors. The binding of the HcR4 ligand to Fc ⁇ R was determined using the receptor binding assay described in Example 6. HcR4 was incubated with immobilized receptors at the indicated concentrations. Plates were washed and bound ligand was detected as described. Results shown are from a representative assay of three performed.
  • HcR4 and HcmR4 increased He-specific antibody responses in high responder SJL mice.
  • SJL mice were immunized with a single 1.0 ug or 0.5 ug dose of Hc, HcR4, or HcmR4. Serum was collected 14 days after immunization and the Hc- specific antibody titers were determined.
  • HcmR4 and HcR4 induced higher antibody responses than Hc alone at both the 1.0 ug and 0.5 ug doses.
  • HcR4 leads to greater induction of secondary T cell responses to recall antigens.
  • LN cells were isolated from SJL mice 14 days post immunization with Hc. LN cells were challenged in vitro with Hc, HcR4, or HcmR4 as indicated.
  • FIG 18. Intranasal delivery of HcR4 results in large and rapid He-specific antibody responses: SJL mice (n 5) received 25 ⁇ g of HcR4 in 10 ⁇ L PBS instilled into each nostril on days 0, 7, and 14. Serum was obtained at days 21 and 28 and Hc specific IgG titers were determined. DETAILED DESCRIPTION
  • the present invention concerns inventive polypeptides.
  • the present invention also concerns compositions and vaccines comprising the inventive polypeptides.
  • This invention describes vaccines and methods of inducing immunity against an antigen using inventive polypeptides described herein.
  • the present invention provides for improved vaccine efficacy by targeting Ag to Fc ⁇ receptors using multiple copies of HCH2 of a human IgG.
  • the vaccines of the present invention can be applied, for example, in the induction of immunity to pathogens, toxins, and peptides expressed by tumor cells.
  • the inventive polypeptides are provided to a subject. Still other embodiments include methods for making the inventive polypeptides and nucleic acids used to encode the inventive polypeptides.
  • Antibodies comprise a large family of glycoproteins with common structural features.
  • An antibody is comprised of four polypeptides that form a three dimensional structure which resembles the letter Y.
  • an antibody is comprised of two different polypeptides, termed the heavy chain and the light chain.
  • An antibody molecule typically consists of three functional regions: the Fc, Fab, and antigen-binding site.
  • the Fc region is located at the base of the Y.
  • the arms of the Y comprise the Fab region.
  • the antigen-binding site is located at the end of each arm of the Y.
  • the area at the fulcrum of the arms of the Y is the hinge region.
  • heavy chain polypeptides There are five different types of heavy chain polypeptides designated as ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ . There are two different types of light chain polypeptides designated K and ⁇ .
  • An antibody typically contains only one type of heavy chain and only one type of light chain, although any light chain can associate with any heavy chain.
  • Antibody molecules are categorized into five classes, IgG, IgM, IgA, IgE, and IgD.
  • the IgG class is further divided into subclasses including IgGl, IgG2, IgG3, and IgG4 for human IgG.
  • An antibody molecule is comprised of one or more Y-units, each Y comprising two heavy chains and two light chains.
  • IgG consists of a single Y-unit.
  • IgM is comprised of 5 Y-like units. Amino acids toward the carboxyl terminal of each heavy chain polypeptide make up a constant region. Amino acids toward the amino terminal of each heavy and light chain polypeptide make up a variable (V) region.
  • variable region hypervariable regions known as complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • One heavy chain and one light chain associate to form an antigen-binding site.
  • Each heavy chain and each light chain includes three CDRs.
  • the six CDRs of an antigen-binding site define the amino acid residues that form the actual binding site for the antigen.
  • CDR variability accounts for the diversity of antigen recognition.
  • the mature human IgGl heavy (H) chain can span 447 amino acid residues.
  • the Fc region of the H chain is essentially the same for all IgGl heavy chain molecules.
  • the Fc region is the portion of the IgGl polypeptide that interacts with Fc receptors.
  • the Fc region can be further subdivided into three consecutive parts, the hinge region, the CH2 domain, and the CH3 domain.
  • the binding site for Fc receptors is found within the hinge and CH2 (HCH2) region.
  • the HCH2 region encompasses amino acid residues 216 to 340 of the human IgGl H chain. (Eu numbering).
  • the hinge region spans residues 216 to 237 whereas the CH2 domain encompasses residues 238 to 340.
  • a recombinant immunoglobulin fusion protein can have an amino-terminus composed of a ligand-binding domain fused to a carboxyl-terminus composed of the hinge, C H 2, and C H 3 regions of Ig.
  • the Ig class sometimes used is IgGl.
  • the hinge, C H 2, and C H 3 regions of IgG are collectively referred to as the Fc region of IgG.
  • the hinge region can provide a flexible linker between the Fc region and the ligand binding domain. It also is the site of inter-chain disulphide bond formation, i.e., the covalent linking of one antibody amino acid chain to another to make the familiar dimeric structure.
  • the hinge region (e.g., the part nearest to the CH2 domain, known as the hinge proximal region) is associated with molecular recognition and binding to Fc ⁇ receptors and complement components.
  • some recombinant immunoglobulin fusion proteins are similar to Ig but lack the variable regions and the CHl domain, which have been replaced by the ligand-binding domain.
  • the recombinant molecule is generated at the cDNA level using recombinant DNA techniques and expressed in cell culture.
  • the recombinant immunoglobulin fusion protein is a disulfide-linked homodimer.
  • fusion partners have been placed at the amino-terminus, such as ligands, enzymes, and peptide epitopes.
  • amino acid chain includes a linear chain of amino acids.
  • the amino acid chain can be chemically or biochemically modified (such as, but not limited to, glycosylation or phosphorylation) or derivatized amino acids, and can have a modified peptide backbone.
  • polypeptide refers to a polymeric form of amino acids of any length, and includes chemically or biochemically modified or derivatized amino acids, as well as amino acid chains having modified peptide backbones.
  • the term includes amino acid chains that are linked, for example, by one or more disulfide bonds, proteins, amino acid chains, saccharides, or polysaccharides.
  • a “fragment” of a polypeptide or protein refers to a polypeptide that is shorter than the reference polypeptide or protein, but that can retain a biological function or activity that is recognized to be the same as the reference polypeptide or protein. Such an activity may include, for example, the ability to stimulate an immune response.
  • a fragment may retain at least one epitope of the reference polypeptide or protein.
  • the shorter polypeptide may retain all or part of a modification (e.g., by glycosylation or phosphorylation) of the reference polypeptide or protein.
  • “Immunological framework” refers to a molecule that comprises two arms attached to an Fc region. The Fc region has the primary structural components of an antibody Fc region, but the arms can be comprised of any molecule and thus are not limited to the Fab-antigen structures of an antibody.
  • the inventive polypeptide can consist of two amino acid chains where each amino acid chain consists of (a) an Fc portion which includes the C-terminus of the amino acid chain; (b) a polymer portion consisting of two to six linear copies of an HCH2 monomer; and (c) an antigen portion which includes the N-terminus of the amino acid chain.
  • the N-terminus of the Fc portion is linked to the C-terminus of the polymer portion, and the N-terminus of the polymer portion is linked to the C- terminus of the antigen portion.
  • the two amino acid chains are linked using one or more disulfide bonds located in the Fc portion of each amino acid chain.
  • the HCH2 monomer can consist of at least a fragment of an HCH2 region, wherein the at least fragment of an HCH2 region includes a hinge region. In some embodiments, at least one hinge region cysteine of the HCH2 monomer is mutated to serine.
  • the Fc portion can comprise, for example, SEQ ID NO: 47 or SEQ ID NO: 48.
  • the inventive polypeptide has an immunoglobulin framework consisting of an Fc region consisting of two amino acid chains wherein each amino acid chain is linked to an arm. Each arm can consist of an HCH2 polymer linked to an antigen portion.
  • the HCH2 polymer can consist of two to six linear copies of an HCH2 monomer, which consists of at least a fragment of an HCH2 region.
  • At least one hinge region cysteine of the HCH2 monomer is mutated to serine, or another non-cysteine amino acid. Sometimes all the hinge region cysteines are mutated.
  • the Fc region comprises the linked Fc portions.
  • the inventive polypeptide can, for example, bind to Fc ⁇ R, target cells expressing Fc ⁇ R, or complement components.
  • the Fc region can be selected or derived from any animal, mammalian, mouse, or human antibody.
  • the Fc region can combine polypeptides of Fc regions from IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgM, IgE, IgG2a, or fragments thereof.
  • the combined polypeptides are identical.
  • Some embodiments of the fragments include fragments comprising a hinge region, a CH2 domain, and a CH3 domain.
  • Exemplary embodiments of sequences that can be used to form the amino acid chain of the Fc region can include, but are not limited to SEQ ID NO: 47 and SEQ ID NO: 48.
  • Linkers can include, but are not limited to, amino acid chains, disulfide bonds, saccharides, polysaccarides, or any known linkers. For example, amino acid chains up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, or 20 amino acids can be used as linkers.
  • the HCH2 monomer can be selected or derived from an Fc region of any animal, mammalian, mouse, or human antibody. It can be a polypeptide from the Fc regions of, for example, but not limited to, IgGl, IgG2, IgG3, IgG4, IgA, IgD, IgM, IgE, IgG2a, or fragments thereof.
  • the HCH2 region can be selected from the group consisting of: a human IgGl HCH2 region, a human IgG2 HCH2 region, a human IgG3 HCH2 region, a human IgG4 HCH2 region, a mouse IgG2a region, and fragments thereof.
  • Exemplary embodiments of sequences that can be used in an HCH2 monomer include, but are not limited to, SEQ ID NO: 9, SEQ ID NO: 10, SEQ YD NO: 51, and SEQ ID NO: 52.
  • the HCH2 polymer can be made from, for example, 1, 2, 3, 4, 5, 6, 7, or 8 linear copies of an HCH2 monomer.
  • the antigen portion includes, but is not limited to, antigens, polypeptides, proteins protein fragments, or any combination thereof.
  • the antigen portion can include proteins or protein fragments linked together in a serial fashion, such as PLP linked to PLP, a fragment of PLP linked to PLP, a fragment of PLP linked to another fragment of PLP, a fragment of PLP linked to a fragment of MBP, MBP linked to a fragment of PLP.
  • the antigen portion is a Botulinum neurotoxin protein, including for example, BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/A Hc, BoNT/A HcN, BoNT/A HcC, or portions, fragments, or variants thereof.
  • a Botulinum neurotoxin protein including for example, BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/A Hc, BoNT/A HcN, BoNT/A HcC, or portions, fragments, or variants thereof.
  • the antigen portion can contain at least one antigenic domain or epitope of an infectious agent, microorganism, tumor antigen, or self protein, including for example, cancer antigens (such as, sarcoma, lymphoma, leukemia, melanoma, carcinoma of the breast, colon carcinoma, carcinoma of the lung, glioblastoma, astrocytoma, carcinoma of the cervix, uterine carcinoma, carcinoma of the prostate, ovarian carcinoma, or portions, fragments, or variants thereof), antigenic domains of infectious agents, antigenic domains of viruses (such as, papilloma virus, Epstein Barr virus, herpes virus, retrovirus, hepatitis virus, influenza virus, herpes zoster virus, herpes simplex virus, human immunodeficiency virus 1, human immunodeficiency virus 2, adenovirus, cytomegalovirus, respiratory syncytial virus, rhinovirus, or portions or variants thereof), antigenic domains of a bacteria (such as,
  • antigen portions include, for example, HSA, HSAl (HSA domain 1), HSA2 (HSA domain 2), HS A3 (HSA domain 3), Fatty Acid Binding Proteins (FABP) such as FABPl, FABP2, FABP3, FABP4, FABP5, FABP6, FABP7, FABP8, FABP9 including FABP5-like 1-7; other examples of antigen portions can be found throughout the application.
  • HSA HSAl
  • HSA2 HSA domain 2
  • HS A3 HSA domain 3
  • Fatty Acid Binding Proteins FABP
  • FABP Fatty Acid Binding Proteins
  • the immunoglobulin Fc region appears to provide some features of the IgG fusion proteins such as stability, covalent dimerization, single-step purification, and ease of detection.
  • the intervening HCH2 polymer appears to confer increased effector function, including, for example, targeting to subsets of cells expressing Fc ⁇ R, increased capacity to ligate Fc ⁇ R, and to bind complement components.
  • the amino-terminal domain can deliver a second signal.
  • multiple molecular signals can be integrated into a single molecule with the potential for synergistic interaction between the domains.
  • the inventive polypeptide comprises multiple HCH2 regions.
  • the polymers were developed using a cloning system that can result in the rapid addition of HCH2 units into a human IgGi Fc region expression vector.
  • Each HCH2 region can be composed of the hinge and CH 2 domain from an Ig such as IgGi, which encompasses the region that can bind Fc ⁇ R and complement.
  • hinge region cysteines of the HCH2 monomer unit were mutated to serines. These mutations can leave intact those hinge residues that interact with FcR and complement.
  • the hinge within the Fc vector was not mutated thus retaining the dimeric structure of IgG.
  • Several unique restriction sites on the 5' end can allow for the directional cloning of amino-terminal domains into the polymer expression constructs.
  • the entirety of the HCH2 region it is not necessary for the entirety of the HCH2 region to be employed in making the HCH2 monomer.
  • the entire human IgGl HCH2 encompasses amino acid residues 216 to 340 of the human IgGl H chain (Eu numbering), with the hinge region spaning residues 216 to 237 and the CH2 domain encompassing residues 238 to 340.
  • the interactions between IgG and Fc receptors have been analyzed in biochemical and structural studies using wild type and mutated Fc.
  • HCH2 polymers were constructed using the human IgGl HCH2 region that encompasses amino acid residues 216 to 340 of the human IgGl H chain. This region contains the sequences that may contribute to Fc receptor binding as well as additional flanking residues. The flanking residues provide structural stability and spacing between the HCH2 regions.
  • HCH2 polymers comprised of fragments within the HCH2 region instead of the entire HCH2 region. This may be done for example to reduce the size of the HCH2 monomer and hence the HCH2 polymer.
  • One way that this could be achieved is through the deletion of flanking residues on either side of the region that has been identified with Fc receptor binding.
  • the hinge could be truncated to span residues 233 to 237 instead of residues 216 to 237 as used in the examples presented herein. Similar considerations apply to the CH2 region that spans residues 238-340 and to the hinge and CH2 regions of other Ig's including IgA, IgD, IgG2, IgG3, IgG4, and IgE. Other embodiments include different configurations of portions of HCH2 regions.
  • the HCH2 polymers can bind to low affinity FcR. In some instances the HCH2 polymers can bind the high affinity FcR receptors, for instance the Fc ⁇ RI receptor. This is a natural consequence of the high binding affinity of the high affinity FcR receptors for the HCH2 region.
  • HCH2 polymers that bind all forms of the low affinity Fc ⁇ R receptors such as, for example, Fc ⁇ RIIa, Fc ⁇ RIIb, Fc ⁇ RIIc, Fc ⁇ RIIIa, and Fc ⁇ RIIIb.
  • the number and spacing of HCH2 monomers comprising the polymer are varied to increase the binding to one type of FcR receptor or conversely to decrease binding to another type of FcR receptor.
  • alterations to the HCH2 monomer can be made to increase specificity of the polymer for one type of Fc ⁇ R receptor or to decrease specific binding to another type of Fc ⁇ R receptor.
  • Such alterations are achieved by mutating certain amino acid residues within the HCH2 sequence to other amino acid residues.
  • the choice of residues to mutate within the HCH2 unit can be informed by choice of target receptor specificity.
  • the specific binding of the HCH2 polymers to different Fc ⁇ R receptors can be enhanced by the presence of and type of glycosylation of the HCH2 polymer.
  • Choice of expression system in which to produce the HCH2 polymers in part determines the extent and type of glycosylation. In the examples presented herein the HCH2 polymers were constructed using
  • HCH2 polymers comprised solely of human sequences to use as immunotherapeutic agents in humans.
  • the polymers are assembled from sequences of other Ig' s including IgA, IgD, IgG, IgM, and IgE.
  • the HCH2 polymers are assembled from sequences of more than one type of Ig, for example a polymer containing HCH2 monomers derived from IgG sequences are linked to HCH2 monomers derived from IgE sequences.
  • the HCH2 polymers are comprised of non-human sequences. The choice of sequences can be determined by the target receptor and host identity (human or non-human).
  • the hinge region cysteines are mutated to amino acid residues other than serine.
  • the HCH2 monomer may be altered or mutated to bind complement components and not to bind to FcR. In other embodiments the HCH2 monomer may be altered or mutated to bind FcR and to not bind complement.
  • the HCH2 polymers were constructed using DNA sequences from human IgGl.
  • the expressed proteins have been evaluated for their interactions with low affinity Fc ⁇ R receptors.
  • the HCH2 polymers are assembled from sequences of other Ig's including IgA, IgD, IgG, IgM, and IgE and these polymers may bind to and interact with the FcR for other Ig's including Fc ⁇ R, Fc ⁇ R, Fc ⁇ R, Fc ⁇ R, and FcRn.
  • the polymers are assembled from sequences of more than one type of Ig, for example a polymer protein containing HCH2 units derived from IgG sequences and IgE sequences will interact and bind with the FcR for more than one type of Ig.
  • HCH2 polymers are constructed from HCH2 monomers consisting of full length HCH2 regions. In some embodiments it may be advantageous to construct HCH2 polymers that contain HCH2 monomers that are smaller than full length HCH2 regions. HCH2 polymers derived from a smaller HCH2 monomer would have a smaller size and mass and yet still retain the ability to effectively bind to and activate FcR or complement. The reduction in the size of the HCH2 monomer is achieved by the removal of sequences that have diminished involvement in the binding to FcR or complement. In some embodiments, the identities of these sequences are known, as are the methods for their removal from the HCH2 monomer unit. The removal of these sequences could maintain the desired binding but yield a polymer of smaller mass.
  • Recombinant HCH2 polymer constructs can mimic the biological activity and functions of immune complexes (ICs), of aggregated IgG (AIG), and of aggregated Fc.
  • ICs immune complexes
  • AIG aggregated IgG
  • Fc aggregated Fc
  • the use of recombinant HCH2 polymer construct can offer several advantages over AIG or Fc aggregates.
  • the number and construction of HCH2 monomers can be altered to hone interaction with FcR' s. Aggregates are by nature heterogeneous with considerable variation between batches whereas inventive polypeptides are precisely defined.
  • the receptors can be specifically activated with constructs containing different numbers of HCH2 monomers. As shown herein, the number of repeating HCH2 monomers available to bind receptor can influence cell function. Cell function can be changed with insertion of additional HCH2 monomers.
  • the constructs of the present invention allow for the measurement of change in receptor function based on IC size. The number of repeating HCH2 monomers included within the polymer construct is variable and can be selected to optimize biological activity. In one embodiment the
  • HCH2 polymers are assembled as disulfide-linked homodimers.
  • the HCH2 polymers are assembled as monomers (e.g., a single chain polypeptide of HCH2 monomers), or hetero- or homo-multimers, and particularly as dimers, tetramers, and pentamers.
  • proteins or protein fragments are found throughout the application and include, for example, ligand-binding domains, extracellular domains of receptors, enzymes, adhesion molecules, cytokines, peptide hormones, immunoglobulin fragments (Fab'), ligands, antigens, and fragments thereof.
  • the site of the fusion of the protein or protein fragment connecting it to the linker or HCH2 polymer may be selected to optimize biological activity, stability, secretion, avidity, and binding specificity.
  • HCH2 polymers using IgGl were designed using sequence data from the human
  • HCH2 polymers composed of the HCH2 polymer region unfused to additional protein domains or Fc or framework sequences.
  • the inventive polypeptides are produced by the insertion of the HCH2 polymeric region into an existing antibody sequence or the sequence of a recombinant protein.
  • This process can be advantageous because of its simplicity.
  • the HCH2 polymeric region is a discrete, modular DNA element designed for easy transfer from one cDNA construct to another.
  • a modular DNA element is sometimes referred to as a 'cloning cassette.
  • the HCH2 polymeric region can be used as a cloning cassette and simply spliced into the existing cDNA for any protein, thus removing several steps from the formation process. In certain circumstances the precise site of insertion within a protein sequence can be determined by experimentation. Using the approach presented in this application, existing monoclonal antibodies and recombinant immunoglobulin fusion proteins can be modified through the addition of the HCH2 polymer region.
  • any of the mentioned polypeptides that are used to construct the inventive polypeptide or parts or portions thereof, can be made from polypeptides that are substantially similar (as defined herein) to the mentioned polypeptides.
  • the inventive polypeptide may be an ingredient or component of a composition, including, for example, a vaccine, emulsion, solution, pill, or any other liquid or solid composition that may be administered to any organism including, for example, plant, animal, mammal, mouse or human.
  • the inventive polypeptide may be glycosylated or free from glycosylation.
  • the invention includes methods for producing an inventive polypeptide comprising (1) preparing a vector comprising the nucleic acid sequence encoding the polypeptide; (2) transfecting a host cell with the vector; (3) culturing the host cell to provide expression; and (4) recovering the polypeptide.
  • the vector can be prepared by any known method, including but not limited to cDNA obtained from reverse transcription, de novo gene synthesis, or obtaining a cDNA template from government or commercial sources.
  • RNA from a cell that expresses the immunoglobulin heavy chain.
  • the RNA can then be used to produce a cDNA using reverse transcription.
  • An example of an expressing cell would be a cell line expressing an antibody of the Ig class of interest. Many monoclonal antibodies are expressed in SPO mouse myeloma cells. Another example is a myeloma cell line of any species that has aberrant expression of Ig heavy chains.
  • ARH-77 (ATCC #: CRL-162) is an example of a human myeloma cell line that produces IgGl heavy chains.
  • cDNA sequences can be built up from smaller DNA sequences, such as oligonucleotides.
  • the advantage of de novo synthesis is that it can provide complete control over the design of the sequences employed to construct the cDNA. This strategy can permit the removal of unwanted restriction sites while introducing others that are more desirable.
  • the codons used in the wild-type gene can be altered to remove a codon bias and thereby improve yields of the expressed protein from the cell of choice.
  • a government source of cDNA template includes obtaining a cDNA clone for the proper Ig type from the IMAGE clone consortium ( ⁇ http://image.llnl.gov/>>).
  • the IMAGE consortium or Integrated Molecular Analysis of Genomes and their Expression Consortium, serves as a repository for mammalian cDNAs for expressed genes.
  • the IMAGE consortium has a full-length cDNA clone for nearly every human and mouse gene.
  • commercial sources such as OpenBiosystems are available.
  • Suitable cells for transfecting and culturing include, but are not limited to insect cells (such as, SF9 cells), mammalian cells (such as, human embryonic kidney cells, HEK 293 cells).
  • Certain embodiments of the present invention involve the use of polypeptides disclosed herein to immunize subjects or as vaccines.
  • immunize or “vaccination” means increasing or activating an immune response against an antigen. It does not require elimination or eradication of a condition but rather contemplates the clinically favorable enhancement of an immune response toward an antigen.
  • the vaccine may be a prophylactic vaccine or a therapeutic vaccine.
  • a prophylactic vaccine comprises one or more epitopes associated with a disorder for which the individual may be at risk (e.g., Botulinum Neurotoxin antigens as a vaccine for prevention of Botulinum intoxication).
  • Therapeutic vaccines comprise one or more epitopes associated with a particular disorder affecting the individual, such as tumor associated antigens in cancer patients.
  • vaccine means an organism or material that contains an antigen in an innocuous form.
  • the vaccine is designed to trigger an immunoprotective response.
  • the vaccine may be recombinant or non-recombinant. When inoculated into a nonimmune host, the vaccine will provoke active immunity to the organism or material, but will not cause disease.
  • Vaccines may take the form, for example, of a toxoid, which is defined as a toxin that has been detoxified but that still retains its major immunogenic determinants; or a killed organism, such as typhoid, cholera and poliomyelitis; or attenuated organisms, that are the live, but non-virulent, forms of pathogens, or it may be antigen encoded by such organism, or it may be a live tumor cell or an antigen present on a tumor cell.
  • a toxoid which is defined as a toxin that has been detoxified but that still retains its major immunogenic determinants
  • a killed organism such as typhoid, cholera and poliomyelitis
  • attenuated organisms that are the live, but non-virulent, forms of pathogens, or it may be antigen encoded by such organism, or it may be a live tumor cell or an antigen present on a tumor cell.
  • Epitope refers to an antigenic determinant of a peptide, polypeptide, or protein; an epitope comprises three or more amino acids in a spatial conformation unique to the epitope. Generally, an epitope consists of at least 5 such amino acids and more usually consists of at least 8 to 10 amino acids. Methods of determining spatial conformation of amino acids include, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance. Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen. Certain embodiments of the present invention pertain to methods of inducing an immune response to an antigen in a subject.
  • antigen means a substance that is recognized and bound specifically by an antibody or by a T cell antigen receptor.
  • Antigens can include polypeptides, peptides, proteins, glycoproteins, polysaccharides, complex carbohydrates, sugars, gangliosides, lipids and phospholipids, fragments thereof, portions thereof and combinations thereof.
  • the antigens can be those found in nature or can be synthetic. Antigens can elicit an antibody response specific for the antigen. Haptens are included within the scope of "antigen.”
  • a hapten is a low molecular weight compound that is not immunogenic by itself but is rendered immunogenic when conjugated with an immunogenic molecule containing antigenic determinants.
  • antigens of the present invention include peptides and polypeptides.
  • the immunogenic polypeptides set forth herein include an antigen polypeptide.
  • Antigen polypeptides that may be used in the immunogenic polypeptides of the present methods include antigens from an animal, a plant, a bacteria, a protozoan, a parasite, a virus, fragments thereof or a combination thereof.
  • An antigen polypeptide is an amino acid sequence that under appropriate conditions results in an immune response in a subject.
  • the immune response may be an antibody response.
  • the antibody response can be measured as an increase in antibody production, as measured by any number of techniques (e.g., ELISA).
  • the immune response may also be a T cell response, such as increased antigen presentation to T cells, or increased proliferation of T cells.
  • the antigen polypeptide may be any polypeptide derived from a virus.
  • the polypeptide may be derived from adenoviridiae (e.g., mastadenovirus and aviadenovirus), herpesviridae (e.g., herpes simplex virus 1, herpes simplex virus 2, Epstein-Barr virus, herpes simplex virus 5, and herpes simplex virus 6), leviviridae (e.g., levivirus, enterobacteria phase MS2, allolevirus), poxyiridae (e.g., chordopoxyirinae, parapoxvirus, avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, molluscipoxvirus, and entomopoxyirinae), papovaviridae (e.g., polyomavirus and papillomavirus), paramyxoviridae (e.g., paramy
  • human immunodeficiency virus 1 and human immunodeficiency virus 2), spumavirus flaviviridae (e.g., hepatitis C virus), hepadnaviridae (e.g., hepatitis B virus), togaviridae (e.g., alphavirus, e.g., Sindbis virus) and rubivirus (e.g., rubella virus), rhabdoviridae (e.g., vesiculovirus, lyssavirus, ephemerovirus, cytorhabdovirus, and necleorhabdovirus), arenaviridae (e.g., arenavirus, lymphocytic choriomeningitis virus, Ippy virus, lassa virus), coronaviridae (e.g., coronavirus and torovirus), influenza virus hemagglutinin (Genbank Accession No.
  • poliovirus I VPl envelope glycoproteins of HIV I, hepatitis B surface antigen, diptheria toxin, streptococcus 24M epitope, gonococcal pilin, pseudorabies virus g50 (gpD), pseudorabies virus II (gpB), pseudorabies virus gill (gpC), pseudorabies virus glycoprotein H, pseudorabies virus glycoprotein E, transmissible gastroenteritis glycoprotein 195, transmissible gastroenteritis matrix protein, swine rotavirus glycoprotein 38, swine parvovirus capsid protein, Serpulina hydodysenteriae protective antigen, bovine viral diarrhea glycoprotein 55, Newcastle disease virus hemagglutinin-neuraminidase, swine flu hemagglutinin, swine flu neuraminidase, foot and mouth disease virus, hog cholera virus, swine influenza virus, African swine fever virus, Mycoplasma
  • Antigen polypeptides useful in the present invention may also be a cancer antigen or a tumor antigen.
  • Any cancer or tumor antigen may be used in accordance with the immunogenic compositions of the invention including, but not limited to, KS 1/4 pan- carcinoma antigen, ovarian carcinoma antigen (CA 125), prostatic acid phosphate, prostate specific antigen, melanoma-associated antigen p97, melanoma antigen gp75, high molecular weight melanoma antigen (HMW-MAA), prostate specific membrane antigen, carcinoembryonic antigen (CEA), polymorphic epithelial mucin antigen, human milk fat globule antigen, colorectal tumor-associated antigens such as: CEA, TAG-72, CO17-1A; GICA 19-9, CTA-I and LEA, Burkitfs lymphoma antigen-38.13, CD19, human B-lymphoma antigen-CD20, CD33, melanoma specific antigen
  • Antigen polypeptides useful in the present invention may also be an autoantigen.
  • Autoantigens known to be associated with autoimmune disease have been described. Included are myelin proteins associated with demyelinating diseases, e.g. multiple sclerosis and experimental autoimmune encephalomyelitis; collagens and rheumatoid arthritis; acetylcholine receptor with myasthenia gravis; insulin, proinsulin, glutamic acid decarboxylase 65 (GAD65), islet cell antigen (ICA512; ICA12) with insulin dependent diabetes.
  • Disease associated myelin proteins include myelin basic protein (MBP), proteolipid protein (PLP), myelin-associated glycoprotein (MAG) and myelin oligodendrocyte glycoprotein (MOG).
  • the antigen polypeptide can be, but is not limited to, abrin, a conotoxin, diacetoxyscirpenol, ricin, saxitoxin, a Shiga-like ribosome inactivating protein, flexal, guanarito, junin, machupo, sabia, tetrodotoxin, a Botulinum neurotoxin, Clostridium perfringens epsilon toxin, a Shigatoxin, Staphylococcal enterotoxin, T-2 toxin, Bovine spongiform encephalopathy agent, epsilon toxin, ricin toxin, Staphylococcal enterotoxin B, or a variant thereof.
  • the antigen polypeptide is administered with the intent of inducing an immune response.
  • the compounds of the present invention can be in various pharmaceutical compositions.
  • the compositions will include a unit dose of the selected polypeptide in combination with a pharmaceutically acceptable carrier and, in addition, can include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, and excipients.
  • “Pharmaceutically acceptable” means a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the fusion protein or other composition without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • any method of preparation of vaccines and immunizing agents can be used, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770.
  • such vaccines are prepared as injectables either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared.
  • the preparation may also be emulsified.
  • the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccines.
  • physiologically acceptable carriers include saline solutions such as normal saline, Ringer's solution, PBS (phosphate-buffered saline), and generally mixtures of various salts including potassium and phosphate salts with or without sugar additives such as glucose.
  • the active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof.
  • Nontoxic auxiliary substances, such as wetting agents, buffers, or emulsifiers may also be added to the composition.
  • Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like.
  • adjuvants are not required for immunization.
  • Sterile injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • the vaccine compositions set forth herein may comprise an adjuvant or a carrier.
  • Adjuvants are any substance whose admixture into the vaccine composition increases or otherwise modifies the immune response to an antigen.
  • Adjuvants can include but are not limited to A1K(SO 4 ) 2 , AlNa(SO 4 ) 2 ,
  • lipid A Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvants, Merck Adjuvant 65, polynucleotides (for example, poly IC and poly AU acids), wax D from Mycobacterium, tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, liposomes or other lipid emulsions, Titermax, ISCOMS, Quil A, ALUN (see U.S. Pat. Nos.
  • TLRs Toll-like receptors
  • LPS Bacterial lipopolysaccharide
  • MPL mono-phosphoryl lipid A
  • TLR5 is expressed on monocytes and DCs and responds to flagellin whereas TLR9 recognizes bacterial DNA containing CpG motifs.
  • Oligonucleotides (OLGs) containing CpG motifs are potent ligands for, and agonists of, TLR9 and have been intensively investigated for their adjuvant properties.
  • cytokines are also useful in vaccination protocols as a result of their lymphocyte regulatory properties.
  • cytokines useful for such purposes are known, including interleukin-12 (IL- 12) that has been shown to enhance the protective effects of vaccines, GM-CSF and IL-18.
  • IL-12 interleukin-12
  • cytokines can be administered in conjunction with antigens and adjuvants to increase the immune response to the antigens.
  • a vaccine composition according to the present invention may comprise more than one different adjuvant.
  • the invention encompasses a therapeutic composition further comprising any adjuvant substance including any of the above or combinations thereof. It is also contemplated that ML-IAP, or one or more fragments thereof, and the adjuvant can be administered separately in any appropriate sequence.
  • the vaccine composition includes a carrier.
  • the carrier may be any suitable carrier, for example a protein or an antigen presenting cell. Examples include serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid.
  • the carrier should be a physiologically acceptable carrier acceptable to humans and safe. Tetanus toxoid or diptheria toxoid are suitable carriers in one embodiment of the invention.
  • the carrier may be dextrans for example sepharose.
  • the timing of administration of the vaccine and the number of doses required for immunization can be determined from standard vaccine administration protocols.
  • a vaccine composition will be administered in two doses. The first dose will be administered at the elected date and a second dose will follow at one month from the first dose.
  • a third dose may be administered if necessary, and desired time intervals for delivery of multiple doses of a particular antigen containing HCH2 polymer can be determined.
  • the antigen containing HCH2 polymer may be given as a single dose. For each recipient, the total vaccine amount necessary can be deduced from protocols for immunization with other vaccines.
  • antigen-HCH2 polymer can vary from subject to subject, depending on the species, age, weight and general condition of the subject, the particular fusion protein used, its mode of administration, and the like. Generally, dosage will approximate that which is typical for the administration of other vaccines, and may be in the range of about 10 ng/kg to 1 mg/kg.
  • Immunizations against toxins and viral infection can be tested using in vitro assays and standard animal models.
  • a mouse can be immunized with a viral antigen polypeptide expressed as a fusion protein with HCH2 polymers and delivered by the methods detailed herein.
  • a blood sample is tested to determine the level of antibodies, termed the antibody titer, using ELISA.
  • the mouse is immunized and, after the appropriate period of time, challenged with the virus to determine if protective immunity against the virus has been achieved.
  • the proper combination of antigen, adjuvant, and other vaccine components can be optimized to boost the immune response.
  • Any known methods for immunization including formulation of a vaccine composition and selection of doses, route of administration and the schedule of administration (e.g. primary and one or more booster doses) can be used (e.g. see Vaccines: From concept to clinic, Paoletti and Mclnnes, eds, CRC Press, 1999).
  • cancer cells human or murine
  • one or more cancer associated antigens can be delivered by the methods described herein.
  • the effect on the cancer cells e.g., reduction of tumor size
  • immunization can include one or more adjuvants or cytokines to boost the immune response.
  • the tests also can be performed in humans, where the end point is to test for the presence of enhanced levels of circulating cytotoxic T lymphocytes against cells bearing the antigen, to test for levels of circulating antibodies against the antigen, to test for the presence of cells expressing the antigen and so forth.
  • the vaccine composition includes antigen presenting cells.
  • the antigen presenting cell can be a dendritic cell (DC).
  • DC may be cultivated ex vivo or derived in culture from peripheral blood progenitor cells (PBPC) and peripheral blood stem cells (PBSC).
  • PBPC peripheral blood progenitor cells
  • PBSC peripheral blood stem cells
  • the dendritic cells may be prepared and used in therapeutic procedures according to any suitable protocol. Different protocols may be adopted to use with patients with different HLA types and different diseases. Incubation of cultured dendritic cells with HCH2 polymers of the invention is envisaged as one means of loading dendritic cells with antigen for subsequent transfer into hosts.
  • peripheral blood progenitor cells PBPC
  • peripheral blood stem cells PBSC
  • PBPC and PBSC can be collected using conventional devices, for example, a Haemonetics.RTM. Model V50 apheresis device (Haemonetics, Braintree, Mass.).
  • MNC mononuclear cells
  • Cells located at the interface between the two phases are withdrawn and resuspended in HBSS.
  • the suspended cells are predominantly mononuclear and a substantial portion of the cell mixture are early stem cells.
  • the stem cells obtained in this manner can be frozen, then stored in the vapor phase of liquid nitrogen. Ten percent dimethylsulfoxide can be used as a cryoprotectant. After all collections from the donor have been made, the stem cells are thawed and pooled. Aliquots containing stem cells, growth medium, such as McCoy's 5A medium, 0.3% agar, and expansion factors (e.g. GM-CSF, IL-3, IL-4, flt3- ligand), are cultured and expanded at 37 0 C. in 5% CO 2 in fully humidified air for 14 days.
  • D The Fc Receptors
  • Fc ⁇ RI CD64
  • AIG and IC bind preferentially to Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD 16), the low affinity receptors for Fc.
  • Fc ⁇ RII and Fc ⁇ RIII are closely related in the structure of their ligand-binding domains.
  • Fc ⁇ RIIA, Fc ⁇ RIIB, and Fc ⁇ RIIC two of which give rise to alternatively spliced variants, code for Fc ⁇ RII.
  • Fc ⁇ RIIa delivers activating signals whereas Fc ⁇ RIIb delivers inhibitory signals.
  • the functional basis for the divergent signals arises from signaling motifs located within the cytoplasmic tails of the receptors.
  • An immunoreceptor tyrosine-based inhibitor motif (ITIM) located in the cytoplasmic tail of the Fc ⁇ RIIb is involved in negative receptor signaling.
  • the ITIM motif is a unique feature of the Fc ⁇ RIIb receptor as it is not apparently present in any other Fc ⁇ receptor class.
  • an activatory immunoreceptor tyrosine-based activation motif or ITAM is located in the cytoplasmic tail of Fc ⁇ RIIa. ITAM motifs transduce activating signals.
  • FcR ⁇ -chains which are identical to the ⁇ -chains of the high affinity IgE receptor (Fc ⁇ RI). While Fc ⁇ RIIa and Fc ⁇ RIIb are widely expressed on myeloid cells and some T-cell subsets they are notably absent from NK cells.
  • Fc ⁇ RIII Human Fc ⁇ RIII is also present in multiple isoforms derived from two distinct genes (Fc ⁇ RIIIA and Fc ⁇ RIIIB).
  • Fc ⁇ RIIIb is unique in its attachment to the cell membrane via a glycosylphosphatidyl anchor. Fc ⁇ RIIIb expression is restricted to neutrophils while Fc ⁇ RIIIa is expressed by macrophages, and NK cells. Fc ⁇ RIIIa is also expressed by some ⁇ T-cell subsets and certain monocytes.
  • Fc ⁇ RIIIa requires the presence of the FcR ⁇ -chain or the CD3 ⁇ -chain for cell surface expression and signal transduction. The FcR ⁇ -chain and the CD3 ⁇ -chain are dimeric and possess ITAM motifs. Fc ⁇ RIIIa forms a multimeric complex with these subunits and signaling is transduced through them. Thus, there is considerable Fc ⁇ R receptor heterogeneity and diverse expression profiles.
  • AIG and IC have been used to target Fc ⁇ RIIIa on immune cells, but as noted earlier production of defined AIG and IC was seen to be problematic. Assembly of complexes by physical or chemical methods is difficult to control with precision resulting in heterogeneity within complexes of similar molecular weight in addition to variations between preparations and changes in composition upon storage. Molecular cloning has been used in the present invention to create molecules that can mimic or approximate AIG and IC function with respect to their interactions with Fc ⁇ R and which allow for the inclusion and targeting of a second protein domain to cells expressing Fc ⁇ R.
  • ICS form These complexes signal through the low affinity IgG receptors that recognize ICS or IgG aggregates preferentially.
  • the low affinity receptors are of two classes Fc ⁇ RII (CD32) and Fc ⁇ RIII (CD16).
  • Fc ⁇ RIIb provides an inhibitory signal for secretion of cytokines that augment immunoglobulin secretion including IgG secretion.
  • FcRIIIa (found on NK cells, monocytes and ⁇ T cells) preferentially recognizes IgGl.
  • One thrust of this invention is directed towards activation of Fc ⁇ RIIIa.
  • Fc ⁇ R The ability of Fc ⁇ R to bind IgG and transmit a signal into the cell depends upon the Fc ⁇ Rs alleles expressed, upon glycosylation, and how the receptor is associated with the signaling subunit. In addition, glycosylation patterns differ between cell types and this too can affect ligand binding to Fc ⁇ RIIIa.
  • Fc ⁇ RIIIa on NK cells is glycosylated with high mannose oligosaccharides, whereas monocyte/macrophage Fc ⁇ RIIIa is not. Perhaps this imparts lower receptor affinity to monocyte/macrophage Fc ⁇ RIIIa relative to NK cell Fc ⁇ RIIIa, adding yet another level of modification to receptor function (Galon et al, 1997; Edberg et al, 1997).
  • Fc ⁇ R function is regulated at several levels, which can have an impact on ligand binding and receptor signaling.
  • IC immune complexes
  • the inventors have initiated studies into the potential immunomodulatory role of immune complexes (IC) in human autoimmune syndromes. Central to these studies are the interactions between IC and FcR.
  • the inventors have used molecular cloning to create molecules that can mimic or approximate IC function with respect to their interactions with FcR and which allow for the inclusion and targeting of a second protein domain to cells expressing FcR.
  • the strategy pursued is to express multiple linear copies of the region of the IgG framework that binds FcR. Expressing only those determinants necessary for FcR engagement and presenting them in a particularly favorable configuration results in novel proteins that are considerably more potent than IC.
  • recombinant IC mimetic proteins described herein will provide both a valuable tool for the examination of IC deposition and in the therapeutic targeting of FcR in autoimmune disorders.
  • the polypeptides include linked antigens.
  • the polypeptides of the invention are used to target an antigen to the cell to enhance the process of internalization and presentation of the antigen by these cells, and ultimately to stimulate an immune response.
  • the polypeptides of the invention specifically bind the antigen directly or bind to epitopes attached to the antigen, e.g., a cloned Fab' fragment covalently attached to the polymer by genetic or chemical means which recognizes the antigen or epitopes attached to the antigen, and targets the bound antigen to antigen presenting cells (APC) for internalization, processing, and presentation.
  • the antigen is linked to the polymers of the invention and at the same time binds a surface receptor of an antigen-presenting cell.
  • the antigen is covalently attached to the polymers of the invention by genetic or chemical means.
  • the polypeptides of this invention can be linked to a cell surface marker.
  • a cell surface marker is a protein, carbohydrate, glycolipid, etc. but most commonly comprises a protein localized to the plasma membrane of a cell having a portion exposed to the extracellular region (e.g. an integral membrane protein or a transmembrane glycoprotein), such that the extracellular portion can be specifically bound by an antibody or other ligand.
  • the term cell surface marker also refers to a polynucleotide sequence encoding such a cell surface protein.
  • cell surface proteins can be used as cell surface markers, such as, for example, a CD (cluster of differentiation) antigen present on cells of hematopoietic lineage (CD2, CD4, CD8, CD21), Gamma-glutamyltranspeptidase, an adhesion protein (ICAM-I, ICAM-2, ELAM- 1, VCAM-I), a hormone, a growth factor, a cytokine receptor, ion channels, and the membrane-bound form of an immunoglobulin chain.
  • CD cluster of differentiation
  • ICAM-2 an adhesion protein
  • ELAM- 1, VCAM-I a hormone
  • growth factor a growth factor
  • cytokine receptor a cytokine receptor
  • ion channels a membrane-bound form of an immunoglobulin chain.
  • Polypeptides that contain HCH2 polymers for use in vaccines.
  • Traditional vaccines consist of killed or attenuated pathogenic organisms or their products administered to develop an immune response.
  • Drawbacks to the traditional approach include unwanted harmful immune responses, inoculation with potentially infectious pathogens, and poor immune responses.
  • these vaccines require coadministration of potent adjuvants to elicit effective antibody responses.
  • Vaccines can be made more effective by delivering those antigenic determinants that are most likely to confer protective immunity.
  • Early attempts to develop peptide based vaccines resulted in poor immune responses due in part to an inefficient presentation of antigen by APCs.
  • APCs capture, internalize and present antigen. In addition they provide important costimulatory signals to T-cells. T-cells, thus activated, are capable of stimulating the production of antibody-forming B cells.
  • Monocytes especially macrophages and dendritic cells, function as APC. Macrophages express all three classes of Fc ⁇ R constitutively whereas dendritic cells express Fc ⁇ RI and Fc ⁇ RII.
  • DCs In contrast mature DCs no longer express Fc ⁇ Rs, become fully active APCs, activate T-cells, and secrete large amounts of IL- 12 (which spurs differentiation of T-cells). DCs are a more potent APC than macrophages though much less numerous.
  • antigenized-antibodies have antigenic determinants grafted into the variable region of IgG. These 'antigenized-antibodies' increased the half-life of antigen and facilitated uptake of antigen by APCs via the Fc ⁇ RI receptor (Zaghouani et al, 1993; Zanetti et al, 1992). Use of antigenized-antibodies have been shown to be more effective at priming antigen specific T-cell responses than peptide alone. Antigenized-antibodies have several limiting features: Since they are directed towards Fc)RI alone, they can be effectively competed against by monomeric serum IgG. Secondly, the design of the molecule limits the size of the antigenizing determinant to a small peptide fragment.
  • antigen has been expressed as a fusion protein with or chemically conjugated to monoclonal antibodies and Fab fragments directed against Fc ⁇ RI and Fc ⁇ RII (Liu et al, 1996b; Guyre et al, 1997).
  • tetanus toxoid epitopes conjugated to anti-Fc ⁇ RI monoclonal antibody one group reported that peptides directed to Fc ⁇ RI were 100 to 1000 fold more efficient than peptide alone in T-cell stimulation (Liu et al, 1996a).
  • use of anti-Fc ⁇ RI Fab' required chemical cross-linking to achieve maximal responses to antigen, thus implicating the low affinity IgG receptors (Keler et al, 2000).
  • inventive polypeptides present antigen to low affinity receptors (Fc ⁇ RII and Fc ⁇ RIII), thus bypassing competition from monomeric serum IgG for binding to Fc ⁇ RI. Additionally there is no need for chemical cross-linking as is necessary when using anti-Fc ⁇ RI Fab'.
  • inventive polypeptides imitate immune complexes. Antigen presented in the context of an immune complex may be a particularly appropriate substrate for APCs. Antigen-linked HCH2 polymers efficiently trigger effector functions that augment the immune response.
  • the inventive polypeptide in tolerance induction can be characterized by the selective lack of an immune response, including lack of a pathogenic immune response to a specific antigen even while leaving other responses of the immune system intact.
  • Immunologic tolerance can be characterized by the selective lack of an immune response, including lack of a pathogenic immune response to a specific antigen even while leaving other responses of the immune system intact.
  • Induction and maintenance of T cell unresponsiveness to a specific antigen may be achieved by several mechanisms that can be broadly summarized as: 1) clonal deletion; 2) anergy; and 3) suppression.
  • Clonal deletion is a process of negative selection whereby T cells with high affinity for self-antigens are deleted in the thymus. Deletion is achieved by programmed cell death (apoptosis).
  • This process of negative selection in the thymus is known as 'central tolerance.
  • Anergy represents a state of immune inactivation characterized by abolished proliferative and cytokine responses. It is induced in cells that previously responded to a given antigen and results in an unresponsive state upon re-stimulation with antigen.
  • TCR T cell receptor
  • IL-IO autocrine inhibitory actions of IL-IO.
  • Suppression of T cell function is a third mechanism by which T cell tolerance can be achieved. Suppression ensues when regulatory T cells are induced to exert "nonspecific" suppressive effects on antigen-specific T cells in their vicinity. This microenvironmental effect is also referred to as 'bystander suppression.
  • B cell tolerance involves concepts and mechanisms similar but not identical to those encountered in T cell tolerance.
  • mature B cells tolerance can be induced through a block in Ig-receptor signaling which results in impaired expression of the B7 costimulatory molecules.
  • Induction of tolerance to either self- or foreign-antigens provides an important therapeutic approach to the treatment of allergies, autoimmune disease and host vs. graft disease (transplant rejection).
  • the therapeutic potential of many foreign biologically active agents is limited by their immunogenicity.
  • Tolerance induction represents one approach for the control of immune responses directed against biologically active foreign agents, thus improving their therapeutic potential.
  • the antigen to be tolerized is presented orally, intradermally, or intravenously.
  • the source of antigen can be in the form of a peptide, a protein, or nucleic acid which can express a peptide or protein.
  • the antigen is then internalized by antigen presenting cells (APC) and presented on the surface of the cell, most typically as a MHC I-antigen complex or as a MHC II -antigen complex.
  • APC antigen presenting cells
  • the inventive polypeptides, which comprise the HCH2 polymers have several advantageous aspects for use as vehicles for tolerance induction.
  • Antigen(s) linked to the HCH2 polymers by chemical or genetic means are targeted to Fc receptors expressed on APC such as macrophages, B cells, and dendritic cells (DC). Fc-receptor-mediated internalization results in processing and presentation of antigen at the cell surface - the key first step in tolerance induction.
  • Macrophages and DC express Fc receptors for both IgG and IgE.
  • HCH2 polymers are expressible which bind both classes of Fc receptor simultaneously - coaggregation of different Fc receptor classes may have advantages over targeting a single class of receptor.
  • Ligation of FcRs induces secretion of ILlO from certain immune cells and, as already noted, ILlO induces anergy in T cells.
  • binding of inventive polypeptides to FcRs may induce a pattern of cytokine secretion that deviates T cell immune responses from a THl type response to a TH2 type response.
  • TH2 type T cells favor the establishment and maintenance of immune tolerance.
  • antigens linked to the HCH2 polymers can promote tolerance induction by both the efficient presentation of antigen to APC and the simultaneous induction of mechanisms that favor establishment of immune tolerance.
  • recombinant monoclonal antibodies can be modified by the introduction of one or more HCH2 units into the Fc region to create an HCH2 polymer of appropriate length within a monoclonal antibody.
  • Monoclonal antibodies modified in this manner will retain their target specificity while acquiring improved or more selective effector function.
  • HCH2 polymers greatly enhance Fc - FcR receptor interactions. More specifically HCH2 polymers of the current invention have greatly improved binding to and enhanced activation of Fc ⁇ RIIIa receptors over that seen with the Fc portion of mAb in current therapeutic use.
  • As enhanced interaction of mAB with Fc ⁇ RIIIa has been documented to have therapeutic benefit in the treatment of malignancies.
  • the inventors envisage modifying existing mAb with the introduction of an HCH2 polymer into the Fc region of the mAb. Monoclonal antibodies with this modification will have enhanced interaction with Fc ⁇ RIIIa receptors.
  • the expression of mAb in mammalian cells typically involves cloning both the H and L gene segments from functional Ig genes into either a single expression vector or separate expression vectors (one for L, one for H genes) that posses the Ig promoter region. Once subcloned the expression vectors possessing the L and H genes are transfected into an appropriate cell line for expression.
  • the use of gene segments insures the presence of intronic sequences, which contain enhancer and other elements that collectively allow for high levels of Ig expression in B cells and myeloma cells. Ig expression systems utilizing the Ig promoter and intronic genetic elements limit protein expression to cells of lymphoid derivation however.
  • Ig expression systems have been developed that use viral promoters and enhancer combinations, such as CMV.
  • the use of viral promoter/enhancer combinations permits strong expression in both lymphoid and non- lymphoid cells lines such as CHO and COS (Norderhaug, et al, 1997).
  • Inclusion of the intronic enhancer from the Ig H gene also directs high level expression in lymphoid cells.
  • H and L gene segments are no longer necessary for efficient expression and can be replaced by their corresponding cDNA's (McLean, et al, 2000).
  • HCH2 polymers into mAb can be achieved by any of several approaches.
  • H chain gene segments within expression vectors are modified by the insertion of the HCH2 polymer cloning cassette into the 5' end of the hinge exon.
  • the modified hinge exon now consists of the HCH2 polymer fused in frame to the hinge sequences.
  • the vector containing the modified H gene is introduced in conjunction with an L gene into an appropriate cell line for mAb expression.
  • Another method is to replace the Fc gene segment with a cDNA segment comprising a splice acceptor signal, the HCH2 polymer fused to an Ig Fc cDNA and a polyA signal.
  • the modified H gene is then transferred into an Ig expression vector capable of directing Ig expression without Ig gene intronic sequences.
  • the vector containing the modified H gene is introduced in conjunction with an L gene into an appropriate cell line for expression.
  • the insertion of HCH2 polymers into mAb expressed from cloned cDNA within expression vectors can also be achieved using similar techniques. For instance, the cDNA encoding the Fc region can be removed from the H chain cDNA and replaced with a DNA segment encoding the HCH2 polymer fused to an Fc cDNA. Conversely, the cDNA encoding the H chain leader, variable and CHl region can be excised and transferred to vectors containing the HCH2 polymer region genetically fused to an Fc cDNA.
  • HCH2 polymers can be introduced into mAb to enhance specificity for other individual FcR receptors, classes of FcR receptors, as blocking reagents for FcR receptors, or for binding to complement factors.
  • Autoimmune diseases are processes in which the immune system mounts an attack against body tissue components. This attack may be mediated by anti-tissue component antibodies produced by B lymphocytes or by cell-mediated tissue destructive processes mediated by T cells, by NK cells, and by monocytes/macrophages. In some autoimmune diseases several tissue damaging mechanisms may operate either concurrently or sequentially.
  • the inventive polypeptides of the current invention can be used in the treatment of autoimmune diseases. They can be used to alter immunity and to deliver therapeutic agents to a delivery site in a patient where the therapeutic agent is effective.
  • autoimmune diseases The number of autoimmune diseases is considerable and some persons may have more than one autoimmune disease. Similarly, signs and symptoms may cover a wide spectrum and severity may also vary widely between afflicted individuals and over time. The reasons why some persons develop autoimmunity while others do not are imperfectly understood but certain recurring themes can be signaled.
  • propensity In many autoimmune processes there is a genetically determined propensity to develop disease. Among the genes that have been linked to propensity to develop autoimmunity are those of the major histocompatibility complex. In addition, environmental factors are thought to play a role. During embryonic development many of those immune system cells that are capable of reacting against self-components are eliminated but some remain so that essentially everyone is at least theoretically capable of mounting an autoimmune response.
  • autoimmune disorders include but are not limited to: systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, Guillain- Barre syndrome, other immune mediated neuropathies including chronic inflammatory demyelinating polyneuropathy, multiple sclerosis and other immune-mediated central nervous system demyelinating diseases, rheumatoid arthritis, Crohn's disease, ulcerative colitis, myasthenia gravis, scleroderma/systemic sclerosis, and dermatomyositis/polymyositis to name some of the more commonly encountered entities.
  • Additional autoimmune diseases include acute glomerulonephritis, nephrotic syndrome, and idiopathic IgA nephropathy among autoimmune processes that affect the kidneys.
  • autoimmune processes that affect the formed elements of the blood are autoimmune aplastic anemia, autoimmune hemolytic anemia, and idiopathic thrombocytopenic purpura.
  • Autoimmune diseases that affect the endocrine organs include Addison's disease, idiopathic hypoparathyroidism, Grave's disease, Hashimoto's thyroiditis, lymphocytic hypophysitis, autoimmune oophoritis, and immunologic infertility in the male.
  • the liver may also be the target of autoimmune processes. Examples include autoimmune hepatitis, hepatitis C virus-associated autoimmunity, immunoallergic reaction drug-induced hepatitis, primary biliary cirrhosis, and primary sclerosing cholangitis.
  • Autoimmune processes of the intestinal tract include pernicious anemia, autoimmune gastritis, celiac disease, Crohn's disease, and ulcerative colitis.
  • Cutaneous autoimmune diseases include dermatitis herpetiformis, epidermolysis bullosa acquisita, alopecia totalis, alopecia areata, vitiligo, linear IgA dermatosis, pemphigus, pemphigoid, psoriasis, herpes gestationis, and cutaneous lupus including neonatal lupus erythematosus.
  • Additional autoimmune diseases with rheumatological features include CREST syndrome, ankylosing spondylitis, Behcet's disease, juvenile rheumatoid arthritis, Sjogren's syndrome, and eosinophilia-myalgia syndrome.
  • Autoimmune diseases can affect the heart. Examples include myocarditis and idiopathic dilated cardiomyopathy, rheumatic fever, Chaga's disease and possibly some components of atherosclerosis.
  • Autoimmune diseases of the central and peripheral nervous systems can occur as a remote effect of malignant tumors. Rarely these same entities occur in the absence of a tumor. Examples include the Lambert-Eaton syndrome, paraneoplastic myelopathy, paraneoplastic cerebellar degeneration, limbic encephalitis, opsoclonus myoclonus, stiff man syndrome, paraneoplastic sensory neuropathy, the POEMS syndrome, dorsal root ganglionitis, and acute panautonomic neuropathy.
  • Autoimmune diseases may affect the visual system. Examples include Mooren's ulcer, uveitis, and Vogt-Koyanagi-Harada syndrome.
  • Other autoimmune processes, or ones in which autoimmunity may contribute to disability include interstitial cystitis, diabetes insipidus, relapsing polychondritis, urticaria, reflex sympathetic dystrophy, and cochleolabyrinthitis.
  • Lymphocytes and macrophages move from the blood into the CNS and attack and destroy myelin and ultimately the myelin forming cells known as oligodendrocytes.
  • the process is one of autoimmunity but the precise target within the CNS against which the immune response is directed remains unknown.
  • There is a genetically determined predisposition to develop multiple sclerosis but there is compelling evidence that environmental factors have a role as well, though the nature of the environmental factors in cause remains unknown.
  • MBP myelin oligodendrocyte protein
  • MOG myelin oligodendrocyte protein
  • the disease may be acute and monophasic in nature, or alternatively chronic, or relapsing-remitting. Affected animals develop flaccid tails, paralysis of the hindlimbs, and incontinence. In severe disease, movement of the forelimbs may also become impaired and animals may become moribund. Histological analysis of the CNS reveals an inflammatory cell infiltrate during the acute stages of disease that may be accompanied by demyelination of the neurons during chronic phases of the disease. EAE is widely used for the study of autoimmune disease and serves as a model for testing potential efficacy of experimental drugs for the treatment of MS and for the treatment of autoimmune diseases in general.
  • the proteins of the current invention were tested for their effect on disease activity in a mouse model of EAE to gain insight into their potential use as therapeutics for the treatment of MS and other autoimmune diseases.
  • Products of the current invention inhibited EAE in the SJL/J mouse.
  • Administration of construct HSAlFc and in particular of HSA 1R4 decreased clinical disease activity during the early acute stages of disease and decreased the frequency of and severity of relapses at later time points as compared to saline-treated controls. Decreased inflammatory cell infiltrates were observed in the CNS of construct-treated animals compared to saline treated-controls.
  • a polynucleotide can be engineered to contain certain sequences that result in (and encode) a biological functional equivalent with more significant changes.
  • Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules, receptors, and such like. So-called “conservative" changes do not disrupt the biological activity of the protein, as the structural change is not one that impinges on the protein's ability to carry out its designated function. It is thus contemplated by the inventors that various changes may be made in the sequence of genes and proteins disclosed herein, while still fulfilling the goals of the present invention.
  • biologically functional equivalents In terms of functional equivalents, it is well understood by the skilled artisan that, inherent in the definition of a "biologically functional equivalent" protein or polynucleotide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule while retaining a molecule with an acceptable level of equivalent biological activity, such as binding to Fc ⁇ Rs. Biologically functional equivalents are thus defined herein as those proteins (and polynucleotides) in which selected amino acids (or codons) may be substituted. In general, the shorter the length of the molecule, the fewer the changes that can be made within the molecule while retaining function. Longer domains may have an intermediate number of changes. The full-length protein will have the most tolerance for a larger number of changes. However, it must be appreciated that certain molecules or domains that are highly dependent upon their structure may tolerate little or no modification.
  • Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, or the like.
  • An analysis of the size, shape or type of the amino acid side- chain substituents reveals that arginine, lysine or histidine are all positively charged residues; that alanine, glycine or serine are all of similar size; or that phenylalanine, tryptophan or tyrosine all have a generally similar shape.
  • Hydropathic amino acid index can be used to confer interactive biological function on a protein (Kyte & Doolittle, 1982). In some instances, certain amino acids may be substituted for other amino acids having a similar hydropathic index or score or still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids with hydropathic indices can be within ⁇ 2 or within ⁇ 1, or within ⁇ 0.5.
  • hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 + 1); glutamate (+3.0 + 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 + 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • the substitution of amino acids with hydrophilicity values can be within +2, or within +1, or within +0.5.
  • substantially similar means a variant amino acid sequence that is at least 80% identical to a native amino acid sequence, or at least 90% identical.
  • the percent identity may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG).
  • the GAP program utilizes the alignment method of Needleman and Wunsch (J. MoI. Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981).
  • Some default parameters for the GAP program can include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353 358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps.
  • Variants may comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics.
  • Beta II turns have been mimicked successfully using cyclic L-pentapeptides and those with D-amino acids. (Weisshoff et al., 1999). Also, Johannesson et al. (1999) report on bicyclic tripeptides with reverse turn-inducing properties.
  • Beta-turns permit changed side substituents without having changes in corresponding backbone conformation, and have appropriate termini for incorporation into peptides by standard synthesis procedures.
  • Other types of mimetic turns include reverse and gamma turns.
  • the size of at least one proteinaceous molecule may comprise, but is not limited to, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about
  • an "amino molecule” refers to any amino acid, amino acid derivative, or amino acid mimic as would be known to one of ordinary skill in the art.
  • the residues of the proteinaceous molecule are sequential, without any non-amino molecule interrupting the sequence of amino molecule residues.
  • the sequence may comprise one or more non-amino molecule moieties.
  • the sequence of residues of the proteinaceous molecule may be interrupted by one or more non-amino molecule moieties.
  • proteinaceous composition encompasses amino molecule sequences comprising at least one of the 20 common amino acids in naturally synthesized proteins, or at least one modified or unusual amino acid, including but not limited to those shown in Table 3.
  • the proteinaceous composition comprises at least one protein, polypeptide or peptide.
  • the proteinaceous composition comprises a biocompatible protein, polypeptide or peptide.
  • biocompatible refers to a substance that produces no significant untoward effects when applied to, or administered to, a given organism according to the methods and amounts described herein.
  • Organisms include, but are not limited to, a bovine, a reptilian, an amphibian, a piscine, a rodent, an avian, a canine, a feline, a fungus, a plant, an archebacteria, or a prokaryotic organism, with a selected animal or human subject being sometimes preferred.
  • Proteinaceous compositions may be made by any technique known, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteinaceous compounds from natural sources, or the chemical synthesis of proteinaceous materials.
  • the nucleotide and protein, polypeptide and peptide sequences for various genes have been previously disclosed, and may be found at computerized databases known to those of ordinary skill in the art.
  • One such database is the National Center for Biotechnology Information's Genbank and GenPept databases (http://www.ncbi.nlm.nih.gov/).
  • Genbank and GenPept databases http://www.ncbi.nlm.nih.gov/.
  • the coding regions for these known genes may be amplified or expressed using the techniques disclosed herein or otherwise known.
  • various commercial preparations of proteins, polypeptides and peptides are known.
  • the proteinaceous composition may comprise at least one antibody.
  • antibody is intended to refer broadly to any immunologic binding agent such as IgG, IgM, IgA, IgD, and IgE.
  • IgG or IgM may be preferred because they are the most common antibodies in the physiological situation and because they are most easily made in a laboratory setting.
  • Polypeptide regions of proteinaceous compounds may be linked via a linker group.
  • a linker group is able to join the compound of interest via a biologically- releasable bond, such as a selectively-cleavable linker or amino acid sequence.
  • antibody is used to refer to any antibody-like molecule that has an antigen binding region, and includes antibody fragments such as Fab 1 , Fab, F(ab') 2 , single domain antibodies (DABs), Fv, scFv (single chain Fv), and the like.
  • DABs single domain antibodies
  • Fv single chain Fv
  • scFv single chain Fv
  • any protein, polypeptide, or peptide containing component may be used in the compositions and methods disclosed herein.
  • the proteinaceous material may be biocompatible.
  • Proteins and peptides suitable for use in this invention may be autologous proteins or peptides, although the invention is clearly not limited to the use of such autologous proteins.
  • autologous protein, polypeptide or peptide refers to a protein, polypeptide or peptide that is derived from or obtained from an organism.
  • Organisms that may be used include, but are not limited to, a bovine, a reptilian, an amphibian, a piscine, a rodent, an avian, a canine, a feline, a fungus, a plant, or a prokaryotic organism, with a selected animal or human subject being sometimes being preferred.
  • the "autologous protein, polypeptide or peptide” may then be used as a component of a composition intended for application to the selected animal or human subject. It can be biocompatible (i.e. from mammalian origin for mammals, from human origin for humans, from canine origin for canines, etc.; it is autologous; it is non-allergenic, or it is non-immunogenic).
  • Autoimmune disease often involves both T-cell and B-cell mediated components that may act dependently or independently of one another, simultaneously or sequentially, resulting in a host-damaging disease often characterized by tissue or cell compromise and a loss of one or more bodily functions.
  • Fc receptors and proteins of the complement cascade are often intimately associated with the generation of the autoimmune response, the regulation of the ongoing immune response, and the effector phase of the immune response (i.e. those mechanisms that lead to tissue or cell destruction or damage).
  • the inventive polypeptides through their ability to bind Fc receptors or complement, may influence disease outcome by their impact upon one or more of these areas.
  • inventive polypeptides may favorably alter disease activity by multiple pathways depending on the fusion protein design and type of disease treated.
  • inventive polypeptides may be designed to contain; multiple units of HCH2 regions, or portions thereof, able to bind Fc receptors, multiple units of HCH2 regions able to bind complement components, or both. It is contemplated that the inventive polypeptide design can be modified to maximize potential benefits achieved from its use in treating a specific disease and its composition may vary from one disease to the next. For example, for the treatment of some diseases it may be preferable to retain the Fc receptor binding ability of the fusion proteins but exclude or diminish binding of components of the complement cascade. The obverse may be preferred for the treatment of other diseases.
  • modifications of the inventive polypeptides as described above are applicable to their use in the treatment of neoplasms, the treatment of infections by viruses or other pathogens, the treatment of warts, and the purposeful induction of an immune response directed against a particular antigen or antigens, as for example in a vaccine.
  • Inventive polypeptides able to bind Fc receptors may influence disease outcome through multiple mechanisms including but not limited to blocking Fc receptor accessibility to endogenously produced Ig and immune complexes. Such blockade would be expected to limit self-antigen presentation by antigen presenting cells and to, as a consequence, diminish autoimmune responses. Blockade of Fc receptors may also limit or diminish tissue and cell destruction. Tissue and cell destruction in autoimmune disease can be mediated by Fc receptor-expressing effector cells (monocytes, neutrophils, macrophages, microglia, NK cells, as well as other cell types) that bind self-antigen reactive Ig bound to tissue or cells.
  • Fc receptor-expressing effector cells monocytes, neutrophils, macrophages, microglia, NK cells, as well as other cell types
  • the inventive polypeptides may modify autoimmune disease by activating cells through Fc receptors and thereby altering the secretion of immunomodulators, the expression of specific cell surface markers, or the type or magnitude of specific cell functions.
  • Modulation of protein secretion might include the decreased or increased production of interleukins including but not limited to IL-2, IL-4, IL-10, IL- 12, IL- 18; cytokines including but not limited to TGF ⁇ , TNF ⁇ , TNF ⁇ ; interferons ⁇ , ⁇ , and ⁇ ; growth factors, and products of the arachidonate cascade.
  • Cellular functions that may be altered include cellular cytotoxicity, cell division, and activation state.
  • HMFGl HFMG2 3.15.C3; M3, M8, M24; M18; 67-D-l l; D547Sp, D75P3, H222; Anti- EGF; LR-3; TAl; H59; 10-3D-2; HmABl,2; MBR 1,2,3; 24 17 1; 24 17-2 (3E1-2); F36/22.M7/105; CI l, G3, H7; B6-2; BI l; Cam 17 1; SM3; SM4; C-MuI (566); 4D5 3H4, 7C2, 6E9, 2C4, 7F3, 2Hl 1, 3E8, 5B8, 7D3, 5B8; OC 125; MO v2; DU-PAN-2; 4F 7 /7A 10; DF 3 ; B72-3; ccccccCEA 11; H17-E2; 3 14 A3; FO23C5; from colorectal tumor sites: B72-3; (17-1A) 1083
  • Another means of defining a targetable tumor is in terms of the characteristics of a tumor cell itself, rather than describing the biochemical properties of an antigen expressed by the cell. Accordingly, the ATCC catalogue exemplifies human tumor cell lines that are publicly available (from ATCC Catalogue).
  • Combination immunotherapies include, for example, interleukin-2, monoclonal or bispecif ⁇ c antibodies such as Rituximab, Herceptin (Trastuzumab), mAb Lym-1, mAb ml70, mAb BC8, mAb Anti-Bl (tositumomab), Campath-1H, anti-CEA mAb MN- 14, mAb HuGl-M195, mAb HuM291, mAb 3F8, mAb C225 (cetuximab), anti-Tac mAb (daclizumab), and mAb hLL2 (epratuzumab).
  • interleukin-2 monoclonal or bispecif ⁇ c antibodies
  • Rituximab Herceptin (Trastuzumab)
  • mAb Lym-1 mAb ml70
  • mAb BC8 mAb Anti-Bl (tositumomab)
  • Campath-1H anti-CEA
  • Combination chemotherapies include, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP 16), tamoxifen, taxol, transplatinum, 5- fluorouracil, vincristin, vinblastin and methotrexate or any analog or derivative variant thereof.
  • CDDP cisplatin
  • carboplatin carboplatin
  • procarbazine mechlorethamine
  • cyclophosphamide camptothecin
  • ifosfamide ifosfamide
  • melphalan chlorambucil
  • bisulfan nitrosurea
  • compositions of the present invention comprise an effective amount of one or more inventive polypeptides, therapeutic agents or additional agent dissolved or dispersed in a pharmaceutically acceptable carrier.
  • Aqueous compositions of the present invention comprise an effective amount of the inventive polypeptides, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate.
  • compositions should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologic Standards.
  • the biological material should be dialyzed to remove undesired small molecular weight molecules or lyophilized for more ready formulation into a desired vehicle, where appropriate.
  • the active compounds will then generally be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, intranasal, intralesional, or even intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the intravenous, intramuscular, subcutaneous, intranasal, intralesional, or even intraperitoneal routes.
  • such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
  • nasal solutions or sprays are generally designed to be compatible with the target tissue type.
  • nasal solutions are usually aqueous solutions designed to be administered to the nasal passages in drops or sprays.
  • an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof.
  • a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or combinations of the fore
  • the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.
  • suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids.
  • traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof.
  • suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, or about 1% to about 2%.
  • composition must be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms, such as bacteria and fungi. It will be appreciated that endotoxin contamination should be kept minimally at a safe level, for example, less that 0.5 ng/mg protein.
  • prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.
  • agents delaying absorption such as, for example, aluminum monostearate, gelatin or combinations thereof.
  • the invention also provides for the use of adjuvants as components in an immunogenic composition compatible with the purified proteins to boost the immune response resulting from vaccination.
  • adjuvants can be selected from the group comprising saponins (e.g, GP-OlOO), or derivatives thereof, emulsions alone or in combination with carbohydrates or saponins, and aluminum-based adjuvants
  • the saponins of the invention can be any saponin as described above or saponin-like derivative with hydrophobic regions, especially the strongly polar saponins, primarily the polar triterpensaponins such as the polar acidic bisdesmosides, e.g.
  • saponin may include glycosylated triterpenoid saponins derived from Quillaja Saponaria Molina of Beta Amytin type with 8-11 carbohydrate moieties as described in U.S. Pat. No. 5,679,354.
  • Saponins as defined herein include saponins that may be combined with other materials, such as in an immune stimulating complex ("ISCOM")-like structure as described in U.S. Pat. No. 5,679,354.
  • Saponins also include saponin-like molecules derived from any of the above structures, such as GPI-0100, such as described in U.S. Pat. No. 6,262,029.
  • the saponins of the invention can be amphiphilic natural products derived from the bark of the tree, Quillaja saponaria. They can consist of mixtures of triterpene glycosides with an average molecular weight (Mw) of 2000. In another embodiment of the invention a purified fraction of this mixture is used. M. Kits
  • kits may comprise a suitably aliquoted inventive polypeptide or additional agent compositions of the present invention, whether labeled or unlabeled, as may be used to prepare a standard curve for a detection assay.
  • kits of the present invention comprise an inventive polypeptide, other polypeptide, peptide, inhibitor, gene, vector or other effectors.
  • Such kits will generally contain, in suitable container means, a pharmaceutically acceptable formulation of an inventive polypeptide in a pharmaceutically acceptable formulation.
  • the kit may have a single container means, or it may have distinct container means for each compound.
  • the container means will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which the immunoglobulin fusion protein formulation is placed, preferably, suitably allocated.
  • the kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer or other diluent.
  • Examples 1 , 2, and 3 describe the cloning and construction of the Fc framework region and HCH2 polymer region of the ligands described in this application.
  • This sequence in the mature polypeptide referred to as R4 is 742 amino acids long and is SEQ ID NO: 13.
  • the digest was extracted Ix with PCIA and Ix with CHCl 3 and DNA was recovered by ethanol precipitation. DNA pellets were washed Ix with 75% ethanol, air dried and resuspended in 15 ⁇ L of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The Hindlll and Sail digested PCR products were ligated into like-digested pBSKS+ vector (Stratagene).
  • the ligation reaction contained 50-100 ng of vector, 20-400 ng of insert, 2 ⁇ L of 10x reaction buffer (660 mM Tris-HCl, pH &.5, 50 mM MgCl 2 , 50 mM DTT, 10 mM ATP), 1 ⁇ L of 5 mM ATP, and 5 U T4 DNA ligase in a final volume of 20 ⁇ L.
  • the ligation reaction proceeded overnight at 16°C.
  • the small letters indicate bases used as clamps or spacers.
  • Boldface letters denote the location of restriction sites.
  • This example describes the isolation and construction of a cDNA coding for the hinge and CH2 region (HCH2) used for the construction of the HCH2 polymer.
  • HCH2 region corresponding to amino acid residues 226-350 of IgGl
  • the hinge region within the HCH2 monomer unit was modified using PCR mutagenesis to change the three cysteines that form interchain disulfide bridges between Fc units to serines.
  • the 3' primer, CH2 ⁇ H3 (Table 5), directed the amplification of the CH2 domain and introduced an in- frame 3' NrMl site separated by a 6 nucleotide spacer from a Hindlll site.
  • Clone pFRM- HS was the template for the PCR reactions. PCR reactions conditions were identical to those described in Example 1. PCR reactions were pooled, extracted with phenol: Chloroform to remove the Taq polymerase and the amplified DNA was recovered with sodium acetate precipitation as described for Example 1. Amplified cDNA was dually digested for two hours in a 120 ⁇ L digestion buffer containing 150 U of EcoRl and 150 U Hindlll. The digest was extracted and DNA was recovered by ethanol precipitation as described for Example 1.
  • HSAl was selected as an antigen since it is poorly antigenic in the mouse. For this reason, we chose to use it to show the utility of using the R4 polypeptide to increase immune responses to a weak antigen in a vaccine formulation.
  • results The expressed polymers are stable, secreted, and soluble and are readily concentrated to useful levels.
  • the proteins are glycosylated, as documented by the difference in predicted and observed molecular weights. Yields correlate inversely with protein size and fall in the range of 0.8 to 2.0 ⁇ g/mL of conditioned medium.
  • Mu_FRM3P-S GGCCGCTAGTCGACTCATTTACCCGGAGTCCGGGAGAAG SEQ ID NO : 20
  • RNA was isolated from mouse liver using the method of Chomczynski and Sacchi (1987). First strand cDNA synthesis was primed with 100 pmol random hexamers using 200 U Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA) and 5 ⁇ g of total RNA in a 20 ⁇ L reaction mixture that was 500 ⁇ M in dNTPs (Pharmacia, Piscataway, NJ), 1 U RNasin/ ⁇ L (Promega Corp., Madison, WI), 10 ⁇ M in DTT, and Ix in first strand buffer. Reaction proceeded at 42° C for 50 min.
  • Insect cells are known to express proteins that can have altered carbohydrate moieties. These alterations may weaken binding of complement factor CIq to these proteins. For this reason the binding of CIq to HCH2 polymers expressed in insect cell line SF9 was investigated. An assay examining the binding of CIq to human IgG or to HCH2 polymers expressed in insect cells was undertaken. Various concentrations of human CIq were allowed to bind to monomeric human IgG, to HSAlFc, or to HSA 1R4 previously immobilized onto wells of a 96 well ELISA plate. The extent of CIq binding was detected using a goat anti-human CIq polyclonal antibody.
  • HCH2-polymers HSA1R2, HSA1R3, and HSA1R4 were assayed for their ability to activate NK cells within PBMC isolates and compared to responses achieved using the Ig-fusion protein, HSAlFc.
  • NK cells express both the low affinity IL-2 receptor, and Fc ⁇ RIII (CD 16) (Nagler et al, 1990).
  • Fc ⁇ RIII CD 16
  • NK cells mount a proliferative response to CD 16 ligation. This triggered response was used as a test of the fitness of the recombinant molecules to engage Fc ⁇ R.
  • PBMC Peripheral blood mononuclear cells
  • PBMC Peripheral blood mononuclear cells
  • AIM V defined serum free medium Gibco BRL
  • Recombinant protein stocks were initially prepared in RPMI 1640 (concentration 1 mg/ml).
  • Recombinant protein stocks were diluted in AIM V medium (Fisher Scientific) to achieve the desired final concentrations as indicated in the drawings.
  • PBMC were plated at a final concentration of 2 x 10 6 cells/ml in 96 well flat bottom plates (0.200 ml/well final volume). Cells were incubated for 72 hours in a humidified incubator at 37°C in 5% atmospheric CO 2 .
  • HSA 1R4 in the high responder mouse strain SJL and in the low responder mouse strain C57BL6.
  • SJL mice respond to vaccination with high antibody titers to antigen while C57BL6 mice respond to vaccination with lower antibody titers.
  • SJL mice were injected intravenously with 50 ⁇ g of HSAl, HSAlFc, or HSA1R4, and serum was obtained 14 days later, and Ab responses were assayed by ELISA.
  • Anti- HSA Ab titers were not detected in mice given HSAl alone at the minimum 1:200 serum dilution used as a cutoff (FIG. 9). Mice given HSA1R4 or HSAlFc developed substantial Ab responses to HSA (FIG. 9).
  • HSA specific titers were fourfold higher in mice injected with HSA1R4 than in mice injected with HSAlFc (p ⁇ 0.05). Equal mass weights of the proteins were injected so that the amount of HSAl in HSAlFc was 2.5 times that in HSA 1R4 (FIG. 9). Anti-HSA IgGi and IgG 20 were increased in response to both immunizations indicating activation of both ThI and Th2 type T cells (FIG. 10). These data show that SJL mice injected with HSAl covalently linked to R4 generate greater antibody responses to HSAl than in mice injected with HSAl alone or to HSAlFc. When Ag/Ab complexes are injected intravenously in mice, greater Ab responses are observed than with Ag alone (Wernersson et al, 1999; Wernersson et al, 2000; Getahun et al, 2004).
  • HSA 1R4 Vaccines are typically injected subcutaneously. Accordingly, the efficacy of HSA 1R4 as an Ag delivery agent was assessed.
  • HSA 1R4 was emulsified in Ribi adjuvant.
  • Ribi adjuvant contains monophosphoryl lipid A (MPL) which signals through Toll-like receptor 4 (TLR4) to activate APC maturation and to increase co-stimulatory molecule expression (Ismaili et al, 2002; Martin et al, 2003).
  • Mice were immunized with HSAl alone, HSAlFc, or HSA 1R4 (50 ⁇ g/mouse), and anti-HSA Ab titers determined in sera obtained 14 days later.
  • MPL monophosphoryl lipid A
  • TLR4 Toll-like receptor 4
  • HSA 1R4 is a potent Ag delivery vehicle for induction of T cell responses.
  • Mice were immunized with 50 ug of HSA1R4 or HSAlFc in Ribi adjuvant.
  • HSAl -specific splenic T cell proliferative responses were measured 14 days later. Taking a stimulation index (SI) of 3 as indicative of response, T cells from mice given HSA 1R4 responded to a 20 fold lower concentration of HSAl than T cells from mice given HSAlFc (FIG. 12A).
  • T cells from mice given HSA1R4 responded better to all 6 concentrations of HSAl tested than T cells from mice given HSAlFc (p ⁇ 0.004) (FIG. 12A).
  • HSA1R4 presents Ag to T cells more efficiently than HSAlFc (FIG 12B).
  • Example 15 Design and construction ofHCH2 polymers for delivery of Botulinum neurotoxin subtype A antigens (BoNT/A).
  • HCH2 polymers that deliver Hc region from Botulinum neurotoxin subtype A worked as well as HCH2 polymers that delivered domain I of human serum albumin. Thus it is the HCH2 polymer that confers enhanced binding to the low affinity Fc receptors. HCH2 polymers also avidly bind Fc ⁇ RI, the high affinity receptor for the Fc region of IgG
  • Hc-reactive T cells respond more strongly to Hc antigens when APCs are primed with 3.6 x 10 "8 M of HcR4 than when APCs are primed with 3.6 x 10 "8 M of Hc alone (Fig 17A). Similar trends are observed at low dose (1.2 x 10 "8 M) (Fig 17B). The data in Fig 17B support a trend seen in antibody titer data indicating that HcmR4 performs better in mice than HcR4, due to a more favorable interaction between the murine ligand and murine Fc ⁇ receptors.
  • Example 19 - R4 administered nasally induces robust antibody responses to antigen.
  • Antigen transcytosed by vesicular transport or FcRn could then be captured by macrophages and DCs and transported to draining lymph nodes. Hc itself binds to epithelial cells and is transcytosed by them. The Hc contained in HcR4 could contribute to transcytosis. Mucosal administration of Hc results in systemic IgG and IgA titers as well as induction of antigen specific mucosal IgA responses. As is the case for Hc administered in adjuvant SC, immune responses to mucosally administered Hc are modest.
  • the recombinant FABP7-R4 fusion proteins were directed into the secretory pathway by proceeding the FABP7 coding region with the leader sequence from human
  • the phosphorylated, hybridized oligonucleotides were ligated directly into the R4pFastBac expression vector that had been previously prepared by digestion with Eco RI and Xho I.
  • the MBP peptide, VHFFKNIVTPRTP (SEQ ID NO: 40), spans 13 amino acids and was introduced into the R4pFastBac expression vector using a strategy identical to that pursued for the PLP peptide.

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Abstract

La présente invention concerne de nouveaux polypeptides. La présente invention concerne également des compositions et des vaccins comportant les polypeptides selon l'invention. Selon d'autres modes de réalisation, les polypeptides selon l'invention sont administrés à un sujet, utilisés pour vacciner, ou utilisés pour induire l'immunité. D'autres modes de réalisation concernent des procédés de fabrication des polypeptides selon l'invention et d'acides nucléiques utilisés pour coder pour les polypeptides selon l'invention.
PCT/US2008/056066 2007-03-06 2008-03-06 Procédés et compositions mettant en œuvre des protéines hybrides d'immunoglobuline polymériques Ceased WO2008109757A2 (fr)

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EP08731555A EP2164859A4 (fr) 2007-03-06 2008-03-06 Procédés et compositions mettant en oeuvre des protéines hybrides d'immunoglobuline polymériques
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WO2013095973A1 (fr) 2011-12-19 2013-06-27 The Rockefeller University Peptides de liaison à hdc-sign
WO2017096221A1 (fr) * 2015-12-02 2017-06-08 The Rockefeller University Anticorps largement neutralisants anti-vih bispécifiques
WO2020110154A1 (fr) * 2018-11-30 2020-06-04 Bharat Biotech International Limited Vaccin thérapeutique chimérique
US20210347849A1 (en) * 2018-07-24 2021-11-11 Good T Cells, Inc. Composition for Preventing or Treating Immune-Related Diseases
US20230338421A1 (en) * 2020-01-10 2023-10-26 TCR2 Therapeutics Inc. Compositions and methods for autoimmunity regulation

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AU2002250293B2 (en) * 2001-03-09 2007-10-11 Arnason, Barry G. Mr Polymeric immunoglobulin fusion proteins that target low-affinity FCGammaReceptors

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CA2679743A1 (fr) 2008-09-12
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EP2164859A4 (fr) 2012-06-06

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